The invention relates generally to lactacystin and analogues thereof.
Eukaryotic cells contain multiple proteolytic systems, including lysosomal proteases, calpains, ATP-ubiquitin-proteasome dependent pathway, and an ATP-independent nonlysosomal process. The major neutral proteolytic activity in the cytosol and nucleus is the proteasome, a 20S (700 kDa) particle with multiple peptidase activities. The 20S complex is the proteolytic core of a 26S (1500 kDa) complex that degrades or processes ubiquitin-conjugated proteins. Ubquitination marks a protein for hydrolysis by the 26S proteasome complex. Many abnormal or short-lived normal polypeptides are degraded by the ubiquitin-proteasome-dependent pathway. Abnormal peptides include oxidant-damaged proteins (e.g., those having oxidized disulfide bonds), products of premature translational termination (e.g., those having exposed hydrophobic groups which are recognized by the proteasome), and stress-induced denatured or damaged proteins (where stress is induced by, e.g., changes in pH or temperature, or exposure to metals). In addition, some proteins, such as casein, do not required ubquitination to be hydrolyzed by the proteasome.
The proteasome has chymotryptic, tryiptic, and peptidyl-glutamyl peptide hydrolizing activities, i.e., the proteasome can cleave peptides on the carboxyl side of hydrophobic, basic, and acidic residues, respectively.
The invention relates to novel compounds structurally related to lactacystin and lactacystin xcex2-lactone. The invention also relates to pharmaceutical compositions including lactacystin and lactacystin analogs.
One aspect of the invention is a pharmaceutical composition containing a compound having the formula 
wherein Z1 is O, S, SO2, NH, or NRa, Ra being C1-6 alkyl; X1 is O, S, CH2, two singly bonded H, CH(Rb) in the E or Z configuration, or C(Rb)(Rc) in the E or Z configuration, each of Rb and Rc, independently, being C1-6 alkyl, C6-12 aryl, C3-8 cycloalkyl, C3-8 heteroaryl, C3-8 heterocyclic radical, or halogen, X1 being two singly bonded H when Z1 is SO2; Z2 is O, S, NH, NRd, CHR1, or CHOR1 in the (R) or (S) configuration, wherein Rd is C1-6 alkyl and R1 is H, halogen, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, NRdRe (except where Z2 is CHOR1), or the side chain of any naturally occurring xcex1-amino acid, or R1 and R2 taken together are a bivalent moiety, provided that when R1 and R2 are taken together, Z1 is NH or NRa and Z2 is CHR1; Re being H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, or C2-6 alkynyl, and the bivalent moiety forming a C3-8 cycloalkyl, C3-8 heteroaryl, C3-8 heterocyclic radical, or C6-12 aryl, where the H in CHR1 is deleted when R1 and R2 taken together form a C3-8 heteroaryl or C6-12 aryl; R2 is C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, azido, C2-6 alkynyl, halogen, ORf, SRf, NRfRg, xe2x80x94ONRfRg, xe2x80x94NRg(ORf) or NRg(SRf) (each of Rf and Rg, independently, being H, C1-6, alkyl, C1-6 haloalkyl, C2-6 alkenyl, or C2-6 alkynyl), or R1 and R2 taken together are a bivalent moiety, the bivalent moiety forming a C3-8 cycloalkyl, C3-8 heteroaryl, C3-8 heterocyclic radical, or C6-12 aryl, where the H in CHR1 is deleted when R1 and R2 taken together form a C3-8 heteroaryl or C6-12 aryl; A1 is H, the side chain of any naturally occurring xcex1-amino acid, or is of the following formula,
xe2x80x94(CH2)mxe2x80x94Yxe2x80x94(CH2)nxe2x80x94R3X3 
wherein Y is O, S, Cxe2x95x90O, Cxe2x95x90S, xe2x80x94(CHxe2x95x90CH)xe2x80x94, vinylidene, xe2x80x94Cxe2x95x90NORh, xe2x80x94Cxe2x95x90NNRiRixe2x80x2, sulfonyl, methylene, CHX4 in the (R) or (S) configuration, or deleted, X4 being halogen, methyl, halomethyl, ORh, SRh, NRiRixe2x80x2, xe2x80x94NRi(ORh), or xe2x80x94NRi(NRiRixe2x80x2), wherein Rh is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, C1-10 acyl, C1-6 alkylsulfonyl, and C6-10 arylsulfonyl; and each of Ri and Rixe2x80x2, independently is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, and C1-10 acyl; m is 0, 1, 2, or 3, and n is 0, 1, 2, or 3; and R3 is straight chain or branched C1-8 alkylidene, straight chain or branched C1-8 alkylene, C3-10 cycloalkylidene, C3-10 cycloalkylene, phenylene, C6-14 arylalkylidene, C6-14 arylalkylene, or deleted, and X3 is H, hydroxyl, thiol, carboxyl, amino, halogen, (C1-6 alkyl)oxycarbonyl, (C7-14 arylalkyl)oxycarbonyl, or C6-14 aryl; or R3 and X3 taken together are the side chain of any naturally occurring xcex1-amino acid; and LO is an organic moiety having 1 to 25 carbon atoms, 0 to 10 heteroatoms, and 0 to 6 halogen atoms; and a pharmaceutically acceptable carrier.
A second aspect is a pharmaceutical composition comprising a compound having the following formula 
wherein Z1 is O, S, SO2, NH, or NRa, Ra being C1-6 alkyl; X1 is O, S, CH2, two singly bonded H, CH(Rb) in the E or Z configuration, or C(Rb)(Rc) in the E or Z configuration, each of Rb and Rc, independently, being C1-6 alkyl, C6-12 aryl, C3-8 cycloalkyl, C3-8 heteroaryl, C3-8 heterocyclic radical, or halogen, provided that when Z1 is SO2, X1 is two singly bonded H; Z2 is CHR1 in the (R) or (S) configuration, R1 being H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, hydroxyl, halogen, a side chain of a naturally occuring xcex1-amino acid, ORd, SRd, or NRdRe (each of Rd and Re, independently, being H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, or C2-5 alkynyl); Z3 is O, S, NH, or NRj, wherein Rj is C1-6 alkyl; X2 is O or S; and A1 is H, the side chain of any naturally occurring xcex1-amino acid, or is of the following formula,
xe2x80x94(CH2)mxe2x80x94Yxe2x80x94(CH2)nxe2x80x94R3X3 
wherein Y is O, S, Cxe2x95x90O, Cxe2x95x90S, xe2x80x94(CHxe2x95x90CH)xe2x80x94, vinylidene, xe2x80x94Cxe2x95x90NORh, xe2x80x94Cxe2x95x90NNRiRixe2x80x2, sulfonyl, methylene, CHX4 in the (R) or (S) configuration, or deleted, X4 being halogen, methyl, halomethyl, ORh, SRh, NRiRixe2x80x2, xe2x80x94NRi(ORh), or xe2x80x94NRi(NRiRixe2x80x2), wherein Rh is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, C1-10 acyl, C1-6 alkylsulfonyl, and C6-10 arylsulfonyl; and each of Ri and Rixe2x80x2, independently is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, and C1-10 acyl; m is 0, 1, 2, or 3, and n is 0, 1, 2, or 3; R3 is straight chain or branched C1-8 alkylidene, straight chain or branched C1-8 alkylene, C3-10 cycloalkylidene, C3-10 cycloalkylene, phenylene, C6-14 arylalkylidene, C6-14 arylalkylene, or deleted; and X3 is H, hydroxyl, thiol, carboxyl, amino, halogen, (C1-6 alkyl)oxycarbonyl, (C7-14 arylalkyl)oxycarbonyl, or C6-14 aryl; or R3 and X3 taken together are the side chain of any naturally occurring xcex1-amino acid; and a pharmaceutically acceptable carrier.
A third aspect is a pharmaceutical composition comprising a compound having one of the following formulae 
wherein Z1 is NH or NRa, Ra being C1-6 alkyl; X1 is O, S, CH2, two singly bonded H, CH(Rb) in the E or Z configuration, or C(Rb) (Rc) in the E or Z configuration, each of Rb and Rc, independently, being C1-6 alkyl, C6-12 aryl, C3-8 cycloalkyl, C3-8 heteroaryl, C3-8 heterocyclic radical, or halogen; Z2 is O, S, NH, or NRj, wherein Rj is C1-6 alkyl; R1 is in the (R) or (S) configuration, and is H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, hydroxyl, halogen, a side chain of a naturally occurring xcex1-amino acid, ORd, SRd, or NRdRe (each of Rd and Re, independently, being H, C1-6 alkyl, C1-6 haloalkyl, C6-12 aryl, C3-8 cycloalkyl, C3-8 heteroaryl, C3-8 heterocyclic radical, or halogen); X1 is O or S; and A1 is H, the side chain of any naturally occurring xcex1-amino acid, or is of the following formula,
xe2x80x94(CH2)mxe2x80x94Yxe2x80x94(CH2)nxe2x80x94R3X3 
wherein Y is O, S, Cxe2x95x90O, Cxe2x95x90S, xe2x80x94(CHxe2x95x90CH)xe2x80x94, vinylidene, xe2x80x94Cxe2x95x90NORh, xe2x80x94Cxe2x95x90NNRiRixe2x80x2, sulfonyl, methylene, CHX4 in the (R) or (S) configuration, or deleted, X4 being halogen, methyl, halomethyl, ORh, SRh, NRiRixe2x80x2, xe2x80x94NRi(ORh), or xe2x80x94NRi(NRiRixe2x80x2), wherein Rh is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, and C2-6 alkynyl, and each of Ri and Rixe2x80x2, independently is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, and C1-10 acyl; m is 0, 1, 2, or 3, and n is 0, 1, 2, or 3; and R3 is straight chain or branched C1-8 alkylidene, straight chain or branched C1-8 alkylene, C3-10 cycloalkylidene, C3-10 cycloalkylene, phenylene, C6-14 arylalkylidene, C6-14 arylalkylene, or deleted, and X3 is H, hydroxyl, thiol, carboxyl, amino, halogen, (C1-6 alkyl)oxycarbonyl, (C7-14 arylalkyl)oxycarbonyl, or C6-14 aryl; or R3 and X3 taken together are the side chain of any naturally occurring xcex1-amino acid; and a pharmaceutically acceptable carrier.
A fourth aspect is a pharmaceutical composition containing a compound having the following formula 
wherein Z1 is O, S, NH or NRj, Rj being C1-6 alkyl; X1 is O or S; R1 is in the (R) or (S) configuration, and is H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, hydroxyl, halogen, a side chain of a naturally occuring xcex1-amino acid, ORd, SRd, or NRdRe (each of Rd and Re, independently, being H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, or C2-5 alkynyl); R2 is H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, C6-12 aryl, C3-8 heteroaryl, or halogen; X2 is O or S; and A1 is H, the side chain of any naturally occurring xcex1-amino acid, or is of the following formula,
xe2x80x94(CH2)mxe2x80x94Yxe2x80x94(CH2)nxe2x80x94R3X3 
wherein Y is O, S, Cxe2x95x90O, Cxe2x95x90S, xe2x80x94(CHxe2x95x90CH)xe2x80x94, vinylidene, xe2x80x94Cxe2x95x90NORh, xe2x80x94Cxe2x95x90NNRiRixe2x80x2, sulfonyl, methylene, CHX4 in the (R) or (s) configuration, or deleted, X4 being halogen, methyl, halomethyl, ORh, SRh, NRiRixe2x80x2, xe2x80x94NRi(ORh), or xe2x80x94NRi(NRiRixe2x80x2), wherein Rh is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, and C2-6 alkynyl, and each of Ri and Ri, independently is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, and C1-10 acyl; m is 0, 1, 2, or 3, and n is 0, 1, 2, or 3; and R3 is straight chain or branched C1-8 alkylidene, straight chain or branched C1-8 alkylene, C3-10 cycloalkylidene, C3-10 cycloalkylene, phenylene, C6-14 arylalkylidene, C6-14 arylalkylene, or deleted, and X3 is H, hydroxyl, thiol, carboxyl, amino, halogen, (C1-6 alkyl)oxycarbonyl, (C7-14 arylalkyl)-oxycarbonyl, or C6-14 aryl; or R3 and X3 taken together are the side chain of any naturally occurring xcex1-amino acid; and a pharmaceutically acceptable carrier.
A fifth aspect is a pharmaceutical composition comprising a compound having the following formula 
wherein Z1 is O, S, NH or NRj, Rj being C1-6 alkyl; X1 is O or S; R1 is H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, hydroxyl, halogen, a side chain of a naturally occuring xcex1-amino acid, ORd, SRd, or NRdRe (each of Rd and Re, independently, being C1-6 alkyl, C1-6 haloalkyl, C6-12 aryl, C3-8 cycloalkyl, C3-8 heteroaryl, C3-8 hetero-cyclic radical, or halogen); R2 is H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, C6-12 aryl, C3-8 heteroaryl, or halogen; Ra is C1-6 alkyl; and A1 is H, the side chain of any naturally occurring xcex1-amino acid, or is of the following formula,
xe2x80x94(CH2)mxe2x80x94Yxe2x80x94(CH2)nxe2x80x94R3X3 
wherein Y is O, S, Cxe2x95x90O, Cxe2x95x90S, xe2x80x94(CHxe2x95x90CH)xe2x80x94, vinylidene, xe2x80x94Cxe2x95x90NORh, xe2x80x94Cxe2x95x90NNRiRixe2x80x2, sulfonyl, methylene, CHX4 in the (R) or (S) configuration, or deleted, X4 being halogen, methyl, halomethyl, ORh, SRh, NRiRixe2x80x2, xe2x80x94NRi(ORh), or xe2x80x94NRi(NRiRixe2x80x2), wherein Rh is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, and C2-6 alkynyl, and each of Ri and Rixe2x80x2, independently is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, and C1-10 acyl; m is 0, 1, 2, or 3, and n is 0, 1, 2, or 3; and R3 is straight chain or branched C1-8 alkylidene, straight chain or branched C1-8 alkylene, C3-10 cycloalkylidene, C3-10 cycloalkylene, phenylene, C6-14 arylalkylidene, C6-14 arylalkylene, or deleted, and X3 is H, hydroxyl, thiol, carboxyl, amino, halogen, (C1-6 alkyl)oxycarbonyl, (C7-14 arylalkyl)-oxycarbonyl, or C6-14 aryl; or R3 and X3 taken together are the side chain of any naturally occurring xcex1-amino acid; and a pharmaceutically acceptable carrier.
A sixth aspect is a pharmaceutical composition containing a compound having the formula 
wherein X1 is O, S, CH2, two singly bonded H, CH(Rb) in the E or Z configuration, or C(Rb)(Rc) in the Eor Z configuration, each of Rb and Rc, independently, being C1-6 alkyl, C6-12 aryl, C3-8 cycloalkyl, C3-6 heteroaryl, C3-8 heterocyclic radical, or halogen; Z1 is O, S, NH, or NRa, Ra being C1-6 alkyl; R1 is H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, hydroxyl, halogen, a side chain of any naturally occuring xcex1-amino acid, ORd, SRd, or NRdRe (each of Rd and Re, independently, being H, C1-6 alkyl, C1-6 halo-alkyl, C6-12 aryl, C3-8 cycloalkyl, C3-8 heteroaryl, C3-8 heterocyclic radical, or halogen); or R1 and R2 taken together are a bivalent moiety which forms a C3-8 cycloalkyl, C3-8 heteroaryl, C3-8 heterocyclic radical, or C6-12 aryl; R2 is H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, azido, C2-6 alkynyl, halogen, ORf, SRf, NRfRg, xe2x80x94ONRfRg, NRg(ORf), or NRg(SRf) (each of Rf and Rg, independently, being H, C1-6, alkyl, C1-6 haloalkyl, C2-6 alkenyl, or C2-6 alkynyl), or R1 and R2 taken together are a bivalent moiety, the bivalent moiety forming a C3-8 cycloalkyl, C3-8 heteroaryl, C3-8 heterocyclic radical, or C6-12 aryl; X2 is O or S; and A1 is in the (R) or (S) configuration, and each of A1 and A2 is independently H, the side chain of any naturally occurring amino xcex1-acid, or is of the following formula,
xe2x80x94(CH2)mxe2x80x94Yxe2x80x94(CH2)nxe2x80x94R3X3 
wherein Y is O, S, Cxe2x95x90O, Cxe2x95x90S, xe2x80x94(CHxe2x95x90CH)xe2x80x94, vinylidene, xe2x80x94Cxe2x95x90NORh, xe2x80x94Cxe2x95x90NNRiRixe2x80x2, sulfonyl, methylene, CHX4 in the (R) or (S) configuration, or deleted, X4 being halogen, methyl, halomethyl, ORh, SRh, NRiRixe2x80x2, xe2x80x94NRi(ORh), or xe2x80x94NRi(NRiRixe2x80x2), wherein Rh is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, and C2-6 alkynyl, and each of Ri and Ri, independently is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, and C1-10 acyl; m is 0, 1, 2, or 3, and n is 0, 1, 2, or 3; and R3 is straight chain or branched C1-8 alkylidene, straight chain or branched C1-8 alkylene, C3-10 cycloalkylidene, C3-10 cycloalkylene, phenylene, C6-14 arylalkylidene, C6-14 arylalkylene, or deleted, and X3 is H, hydroxyl, thiol, carboxyl, amino, halogen, (C1-6 alkyl)oxycarbonyl, (C7-14 arylalkyl)-oxycarbonyl, or C6-14 aryl; or R3 and X3 taken together are the side chain of any naturally occurring xcex1-amino acid; and a pharmaceutically acceptable carrier.
A seventh aspect is a pharmaceutical composition containing a compound having the following formula 
wherein Z1 is NH or NRa, NRa being C1-6 alkyl;
X1 is O, S, CH2, two singly bonded H, CH(Rb) in the E or Z configuration, or C(Rb)(Rc) in the E or Z configuration, each of Rb and R., independently, being C1-6 alkyl, C6-12 aryl, C3-8 cycloalkyl, C3-8 heteroaryl, C3-8 heterocyclic radical, or halogen;
Z2 is O, S, NH, or NRj, Rj being C1-6 alkyl;
R1 is in the (R) or (S) configuration, and is H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, hydroxyl, halogen, a side chain of any naturally occuring amino acid, ORd, SRd, or NRdRe (each of Rd and Re, independently, being H, C1-6 alkyl, C1-6 haloalkyl, C6-12 aryl, C3-8 cycloalkyl, C3-8 heteroaryl, C3-8 heterocyclic radical, or halogen); or R1 and R2 taken together are a bivalent moiety which forms a C3-8 cycloalkyl, C3-8 heteroaryl, C3-8 heterocyclic radical, or C6-12 aryl; R2 is H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, azido, C2-6 alkynyl, halogen, ORf, SRf, NRfRg, xe2x80x94ONRfRg, xe2x80x94NRg(ORf), or xe2x80x94NRg(SRf) (each of Rf and Rg, independently, being H, C1-6, alkyl, C1-6 haloalkyl, C2-6 alkenyl, or C2-6 alkynyl), or R1 and R2 taken together are a bivalent moiety, the bivalent moiety forming a C3-8 cycloalkyl, C3-8 heteroaryl, C3-8 heterocyclic radical, or C6-12 aryl; X2 is O or S; and each of A1 and A2 is independently in the (R) or (S) configuration, and is independently H, the side chain of any naturally occurring xcex1-amino acid, or is of the following formula,
xe2x80x94(CH2)mxe2x80x94Yxe2x80x94(CH2)nxe2x80x94R3X3 
wherein Y is O, S, Cxe2x95x90O, Cxe2x95x90S, xe2x80x94(CHxe2x95x90CH)xe2x80x94, vinylidene, xe2x80x94Cxe2x95x90NORh, xe2x80x94Cxe2x95x90NNRiRixe2x80x2, sulfonyl, methylene, CHX4 in the (R) or (S) configuration, or deleted, X4 being halogen, methyl, halomethyl, ORh, SRh, NRiRixe2x80x2, xe2x80x94NRi(ORh), or xe2x80x94NRi(NRiRixe2x80x2), wherein Rh is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, and C2-6 alkynyl, and each of Ri and Rixe2x80x2, independently is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, and C1-10 acyl; m is 0, 1, 2, or 3, and n is 0, 1, 2, or 3; and R3 is straight chain or branched C1-8 alkylidene, straight chain or branched C1-8 alkylene, C3-10 cycloalkylidene, C3-10 cycloalkylene, phenylene, C6-14 arylalkylidene, C6-14 arylalkylene, or deleted, and X3 is H, hydroxyl, thiol, carboxyl, amino, halogen, (C1-6 alkyl)oxycarbonyl, (C7-14 arylalkyl)-oxycarbonyl, or C6-14 aryl; or R3 and X3 taken together are the side chain of any naturally occurring xcex1-amino acid; and a pharmaceutically acceptable carrier.
An eighth aspect is a pharmaceutical composition containing a compound of the formula 
wherein Z1 is O, NH, or NRa, NRa being C1-6 alkyl; X1 is O, S, CH2, or two singly bonded H; each of A1 and A2 is independently H, the side chain of any naturally occurring xcex1-amino acid, or is of the following formula,
xe2x80x94(CH2)mxe2x80x94Yxe2x80x94(CH2)nxe2x80x94R3X3 
wherein Y is O, S, Cxe2x95x90O, Cxe2x95x90S, xe2x80x94(CHxe2x95x90CH)xe2x80x94, vinylidene, xe2x80x94Cxe2x95x90NORh, xe2x80x94Cxe2x95x90NNRiRixe2x80x2, sulfonyl, methylene, CHX4 in the (R) or (S) configuration, or deleted, X4 being halogen, methyl, halomethyl, ORh, SRh, NRiRixe2x80x2, xe2x80x94NRi(ORh), or xe2x80x94NRi(NRiRixe2x80x2), wherein Rh is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, and C2-6 alkynyl, and each of Ri and Ri, independently is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, and C1-10 acyl; m is 0, 1, 2, or 3, and n is 0, 1, 2, or 3; and R3 is straight chain or branched C1-8 alkylidene, straight chain or branched C1-8 alkylene, C3-10 cycloalkylidene, C3-10 cycloalkylene, phenylene, C6-14 arylalkylidene, C6-14 arylalkylene, or deleted, and X3 is H, hydroxyl, thiol, carboxyl, amino, halogen, (C1-6 alkyl)oxycarbonyl, (C7-14 arylalkyl)-oxycarbonyl, or C6-14 aryl; or R3 and X3 taken together are the side chain of any naturally occurring xcex1-amino acid; and R12 is H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, or C2-6 alkynyl; and a pharmaceutically acceptable carrier.
A ninth aspect is a pharmaceutical composition comprising a compound having the formula 
wherein Z1 is NH, or NRa, NRa being C1-6 alkyl; each of X1 and X2, independently, is O or S; each of A1 and A2 is independently in the (R) or (S) configuration, and is independently H, the side chain of any naturally occurring amino acid, or is of the following formula,
xe2x80x94(CH2)mxe2x80x94Yxe2x80x94(CH2)nxe2x80x94R3X3 
wherein Y is O, S, Cxe2x95x90O, Cxe2x95x90S, xe2x80x94(CHxe2x95x90CH)xe2x80x94, vinylidene, xe2x80x94Cxe2x95x90NORh, xe2x80x94Cxe2x95x90NNRiRixe2x80x2, sulfonyl, methylene, CHX4 in the (R) or (s) configuration, or deleted, X4 being halogen, methyl, halomethyl, ORh, SRh, NRiRixe2x80x2, xe2x80x94NRi(ORh), or xe2x80x94NRi(NRiRixe2x80x2), wherein Rh is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, and C2-6 alkynyl, and each of Ri and Rixe2x80x2, independently is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, and C1-10 acyl; m is 0, 1, 2, or 3, and n is 0, 1, 2, or 3; and R3 is straight chain or branched C1-8 alkylidene, straight chain or branched C1-8 alkylene, C3-10 cycloalkylidene, C3-10 cycloalkylene, phenylene, C6-14 arylalkylidene, C6-14 arylalkylene, or deleted, and X3 is H, hydroxyl, thiol, carboxyl, amino, halogen, (C1-6 alkyl)oxycarbonyl, (C7-14 arylalkyl)-oxycarbonyl, or C6-14 aryl; or R3 and X3 taken together are the side chain of any naturally occurring xcex1-amino acid; and a pharmaceutically acceptable carrier.
Many of the compounds described above are novel compounds; the novel compounds are also claimed. The invention also encompasses lactacystin analogues that can be made by the synthetic routes described herein, and methods of treating a subject having a condition mediated by proteins processed by the proteasome by administering (e.g., orally) to a subject an effective amount of a pharmaceutical composition disclosed herein. These methods include treatments for cancer, inflammation (e.g., inflammatory responses associated with allergies, bone marrow or solid organ transplantation, or disease states), psoriasis, and restenosis.
The compounds disclosed herein are highly selective for the proteasome, and do not inhibit other proteases such as trypsin, xcex1-chymotrypsin, calpain I, calpain II, papain, and cathepsin B.
Other features or advantages of the present invention will be apparent from the following detailed description, and also from the appending claims.
Terms
The term xe2x80x9cnaturally occurring amino acidxe2x80x9d is meant to include the 20 common xcex1-amino acids (Gly, Ala, Val, Leu, Ile, Ser, Thr, Asp, Asn, Lys, Glu, Gln, Arg, His, Phe, Cys, Trp, Tyr, Met and Pro), and other amino acids that are natural products, such as norleucine, ethylglycine, ornithine, methylbutenylmethylthreonine, and phenylglycine. Examples of amino acid side chains include H (glycine), methyl (alanine), xe2x80x94(CH2xe2x80x94(Cxe2x95x90O)xe2x80x94NH2 (asparagine), xe2x80x94CH2xe2x80x94SH (cysteine), and xe2x80x94CH(OH)CH3 (threonine).
The term xe2x80x9cinhibitorxe2x80x9d is meant to describe a compound that blocks or reduces the activity of an enzyme (e.g. the proteasome, or the X/MB1 subunit or xcex1-chain of the 20S proteasome). An inhibitor may act with competitive, uncompetitive, or noncompetitive inhibition. An inhibitor may bind reversibly or irreversibly, and therefore the term includes compounds which are suicide substrates of an enzyme. An inhibitor may modify one or more sites on or near the active site of the enzyme, or it may cause a conformational change elsewhere on the enzyme. Thus, some compounds disclosed herein (e.g., where LO is an oxiranyl or aldehyde group) react with the enzyme by bonding to the carbon atom corresponding to C4 of lactacystin (e.g., resulting in a C4 having a hydroxyl or thiol), while other compounds react with the enzyme to release a leaving group (e.g., L1) corresponding to an acylation.
An alkyl group is a branched or unbranched hydrocarbon that may be substituted or unsubstituted. Examples of branched alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, sec-pentyl, isopentyl, tert-pentyl, isohexyl. Substituted alkyl groups may have one, two, three or more substituents, which may be the same or different, each replacing a hydrogen atom. Substituents are halogen (e.g., F, Cl, Br, and I), hydroxyl, protected hydroxyl, amino, protected amino, carboxy, protected carboxy, cyano, methylsulfonylamino, alkoxy, acyloxy, nitro, and lower haloalkyl. Similarly, cycloalkyl, aryl, arylalkyl, alkylaryl, heteroaryl, and heterocyclic radical groups may be substituted with one or more of the above substituting groups. Examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. An aryl group is a C6-20 aromatic ring, wherein the ring is made of carbon atoms (e.g., C6-14, C6-10 aryl groups, wherein C8 aryl may be an alkylaryl such as 2,3-dimethylphenyl or an arylalkyl such as phenylethyl, or an alkylarylalkyl such as o-methyl-benzyl). Examples of haloalkyl include fluoromethyl, dichloromethyl, trifluoromethyl, 1,1-difluoroethyl, and 2,2-dibromoethyl.
A heterocyclic radical contains at least one ring structure which contains carbon atoms and at least one heteroatom (e.g., N, O, S, or P). A heteroaryl is an aromatic heterocyclic radical. Examples of heterocyclic radicals and heteroaryl groups include: thiazolyl, thienyl, furyl, 1-isobenzofuranyl, 2H-chromen-3-yl, 2H-pyrrolyl, N-pyrrolyl, imidazolyl, pyrazolyl, isothiazolyl, isooxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyradazinyl, indolizinyl, isoindolyl, indolyl, indazolyl, purinyl, phthalazinyl, cinnolinyl, and pteridinyl. A heterocylic radical may be attached to another moiety via a carbon atom or a heteroatom of the heterocyclic radical.
A (Cn alkyl)oxycarbonyl group has the formula Rxe2x80x94Oxe2x80x94(Cxe2x95x90O)xe2x80x94. (C1-6 alkyl)oxycarbonyl, therefore, includes methoxycarbonyl and hexyloxycarbonyl. A C1-10 acyl group as used herein is of the formula xe2x80x94(Cxe2x95x90O)xe2x80x94L3 and contains 1 to 10 carbon atoms (e.g., C1-6) and 1-5 heteroatoms (at least one oxygen). Examples of such acyl groups include formyl, acetyl, benzyloxycarbonyl, tert-butoxycarbonyl, trifluoroethyloxycarbonyl, thiobenzoyl, phenylamidocarbonyl, and 4-nitrophenoxycarbonyl.
An alkylene is a bivalent radical derived from alkanes by removing two hydrogens from two different carbon atoms. Examples of alkylenes include xe2x80x94CH2xe2x80x94CH(R)xe2x80x94CH2 and 1,4-cyclohexylene. An alkylidene is a bivalent radical derived from alkenes by removing two hydrogens from the same carbon atom, such as 1-propanyl-3-ylidene (xe2x95x90CHxe2x80x94CH2xe2x80x94CH2xe2x80x94).
An aromatic carbon atom, as used herein, is a carbon atom within an aromatic system such as benzene or naphthalene or a heteroaromatic system such as quinoline. Examples of nonaromatic carbon atoms include the carbons atoms in Rxe2x80x94(Cxe2x95x90O)xe2x80x94R, xe2x80x94CH2Cl, xe2x80x94CH2xe2x80x94 and Rxe2x80x94(Cxe2x95x90O)xe2x80x94Oxe2x80x94R. A fragment formula weight is the combined atomic weight of the fragment or moiety indicated. For example, the fragment formula weight of methyl is 15 and the fragment formula weight of hydroxyl is 17.
A leaving group departs from a substrate with the air of electrons of the covalent bond between the leaving group and the substrate; preferred leaving groups stabilize those electrons via the presence of electron withdrawing groups, aromaticity, resonance structures, or a combination thereof. Examples include halide (I and Br are preferred), mesylate, trifluoromethanesulfonate, p-toluenesulfonate, p24-nitrobenzensulfonate, benzoate, p-nitrobenzoate, p-nitrobenzyl, and C2-5 haloalkylcarbonyloxy such as trifluoroacetate.
Numerous thiol-, amino-, hydroxy- and carboxy-protecting groups are well-known to those in the art. In general, the species of protecting group is not critical, provided that it is stable to the conditions of any subsequent reaction(s) on other positions of the compound and can be removed at the appropriate point without adversely affecting the remainder of the molecule. In addition, a protecting group may be substituted for another after substantive synthetic transformations are complete. Clearly, where a compound differs from a compound disclosed herein only in that one or more protecting groups of the disclosed compound has been substituted with a different protecting group, that compound is within the invention. For example, where a compound differs from a compound disclosed herein only in that one or more hydrogens from a thiol-, amino-, or hydroxyl-moiety (or a hydrogen or hydroxyl from a carboxyl moiety) has been substituted with a protecting group to form a carboxyl or sulfonyl ester, that protected compound is within the invention. Sulfonyl esters include alkylsulfonyls (e.g., methylsulfonyl or mesyl) and arylsulfonyls (e.g., tosyl). Further examples and conditions are found in T. W. Greene, Protective Groups in organic Chemistry, (1st ed., 1981; 2nd ed., 1991, T. W. Greene and P. G. M. Wuts).
The invention also encompasses isotopically-labelled counterparts of compounds disclosed herein. An isotopically-labelled compound of the invention has one or more atoms replaced with an isotope having a detectable particle- or x-ray-emitting (radioactive) nucleus or a magnetogyric nucleus. Examples of such nuclei include 2H, 3H, 13C, 15N, 19F, 29Si, 31p, 32p and 125I. Isotopically-labelled compounds of the invention are particularly useful as probes or research tools for spectrometric analyses, radioimmunoassays, binding assays based on xcex3- or xcex2-scintillation, fluorography, autoradiography, and kinetic studies such as inhibition studies or determination of primary and secondary isotope effects.
The following abbreviations are used in the synthetic description:
AIBN, 2,2xe2x80x2-azobis(isobutyronitrile); Bn, benzyl; BOP-Cl, bis(2-oxo-3-oxazolidinyl)phosphinic chloride; Bu2BOTf, dibutylboron triflate; CDI, N,Nxe2x80x2-carbonyl-diimidazole; Cp, cyclopentadienyl; DBU, 1,8-diazabicyclo-[5.4.0]undec-7-ene; DCC dicyclohexylcarbodiimide; DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; DEAD, diethylazodicarboxylate; DIBAL-H diisobutylaluminum hydride; DMF, dimethylformamide; Gilbert reagent, dimethyl diazomethylphosphonate; LDA, lithium diisopropylamide; LiHMDS, lithium hexamethyldisilazamide; mesylate, methanesulfonic acid ester; Mitsunobu reagents, (DEAD, PPh3, and nucleophile); NMO, N-methylmorpholine-N-oxide; Ph, phenyl; PhFl, 9-phenyl-9-fluorenyl; Ph2NTf, N-phenyltrifluoromethanesulfonimide; Swern oxidationreagents ((COCl)2, DMSO, Et3N); TBAF, tetrabutylammonium fluoride; TBS, tert-butyldimethylsilyl; TCDI, thiono-N,Nxe2x80x2-carbonyldiimidazole; Tf2O, trifluoromethane-sulfonic acid anhydride; TMS, trimethylsilyl; triflate, trifluromethane sulfonate ester; and TsCl, p-toluene-sulfonyl chloride.
The following reagents are also used: 
The invention is based, in part, on the structure-function information disclosed herein which suggests the following preferred stereochemical relationships. Note that a preferred compound may have one, two, three, or more stereocenters having the indicated up-down (or xcex2-xcex1 where xcex2 as drawn herein is above the plane of the page) or (R)-(S) relationship. In some embodiments, preferred configurations are 7(R), 9(S), 6(S), or combinations thereof. In lactacystin analogs, R2 and LO preferably have a syn relationship to allow formation of a lactone or lactam-type intermediate, which is believed to be related to the activity of the compounds disclosed herein as proteasome inhibitors. However, it is not required that every stereocenter conform to the structures below. 
A person of skill will recognize that the compounds described herein preserve certain stereochemical and electronic characteristics found in either lactacystin or lactacystin xcex2-lactone. For example, the hydroxyisobutyl group and the configuration of the hydroxyl group on C9 are believed to be important for recognition of the target, as are the configurations of the C6 hydroxyl and the C7 methyl of the xcex3-lactam ring. However, the N-acetylcysteine moiety is not required for activity. Moieties such as R2, R1, and particularly A1 (e.g., where A1 is a side chain of a naturally occurring xcex1-amino acid such as Val, Leu, Lys, and Phe) can be modified to control selectivity for the proteasome, and selectivity for a particular peptidase activity of the proteasome. In combination, these moieties simulate certain peptides or proteins processed or degraded by the proteasome (i.e., are peptidomimetics).
The invention is also based, in part, on the finding that lactacystin and lactacystin xcex2-lactone are highly selective for the X/MB1 subunit and xcex1-chain of the proteasome and do not inhibit the activity of proteases such as trypsin, xcex1-chymotrypsin, calpain I, calpain II, cathepsin, and papain. Such selectivity is useful to formulate a pharmaceutical composition with fewer side effects and to evaluate basic research results involving any subunit of the proteasome in conjunction with the selective inhibition of the X/MB1 subunit and xcex1-chain.
Turning to the novel compounds described by the formulas of compounds contained within the pharmaceutical compositions, several embodiments are next considered.
Embodiments of the first aspect include compounds having one or more of the following limitations. In one limitation L1 is linked by an oxygen or preferably sulfur atom to the carbon atom bonded to X2. Such an L1 forms a thioester, thus providing a good leaving group. The precise nature of the thioester group is generally not critical. In initial experiments, the magnitude of activity measured by Kobs/[I] for both large and small thioesters was about the same, whether the thioester terminus was a simple arylalkyl group or a more complex, branched moiety with intervening amide linkages and terminal carboxylic acid, esters, or aminocarbonyl groups. In one embodiment, Z1 is NH or NRa; X1 is O or S; Z2 is CHR1 or CHOR1, R1 being H, halogen, C1-6 alkyl, or C1-6 haloalkyl; R2 is ORf; and L0 is xe2x80x94(Cxe2x95x90X2)xe2x80x94L1, X2 being O and L1 being linked by a sulfur atom to the carbon atom bonded to X2. In another embodiment, L1 is selected from substituted or unsubstituted C6-20 arylalkylcarbonylthio, C6-20 arylthio, C2-8 alkylcarbonylthio, and C1-12 alkylthio. As defined above in the xe2x80x9cTermsxe2x80x9d section, arylthio includes arylthio, alkylarylthio, arylalkylthio, and alkylarylalkylthio groups.
In other limitations, A1 is C5-20 alkyl when LO is carboxyl or (C1-4 alkyl)oxycarbonyl and A1 is alkyl; only one of A1 and LO is selected from carboxyl and (C1-14 alkyl)oxycarbonyl; A1 cannot be H; L1 has at least 3 carbon atoms when Z1 and Z2 are both NH, and A1 is methoxy; when R1 and R2 are taken together, Z2 is CR1; and when one of A1 and LO is (C1-14 alkyl)oxycarbonyl or carboxyl, the other of A1 and LO has a fragment formula weight of at least 20. Novel compounds of the first, fifth, and seventh aspects generally do not have A1 being H.
Further embodiments of the first aspect, when Z1 is NH and Z2 is (R) CHR1, are compounds wherein: L1 has between 0 and 3 nonaromatic acyclic carbon atoms when there are 5 heteroatoms; L1 has between 6 and 15 nonaromatic acyclic carbon atoms when there is only one oxygen atom; and L1 has 0, 1, 3, or 5 nonaromatic carbon atoms when there are three halogen atoms.
Further embodiments of the first aspect include a compound wherein Z2 is CHR1 and Z1 is NH or NRa; wherein LO is C2-16 oxiranyl; L1 is hydroxy, C1-12 alkoxy, C1-12 alkyl-sulfonyloxy, C6-20 arylsulfonyloxy, C7-20 arylalkyl, C6-20 aryloxy, C6-20 arylalkylcarbonyloxy, C2-8 alkylcarbonyloxy, C2-8 alkylcarbonylthio, C1-12 alkylthio, C6-20 arylalkylcarbonylthio, or C6-20 arylthio; or L2 is H, C1-2 haloalkyl, or C1-6 alkyl. In addition, Z2 is O, S, NH, or NRd. In another embodiment, Z2 is O, S, NHJ, NRd, or CHR1; and Rh is H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, or C2-6 alkynyl.
Further embodiments of the first aspect include compounds wherein Z1 is O, S, or SO2; wherein Z2 is O, S, NH, or NRd; wherein X1 is CH2, CHRb, or C(Rb)(Rc); wherein R2 is C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, or halogen; wherein R2 is ORf, SRf, or NRfRg; wherein Z2 is in the beta (above plane of page) configuration; wherein CHX4 is in the alpha (below plane of paper) configuration; wherein R2 is in the beta configuration; or combinations thereof.
The invention also provides a pharmaceutical composition wherein LO is H or xe2x80x94(Cxe2x95x90X2)xe2x80x94L1, X2 being O and L1 being linked by an oxygen or sulfur atom to the carbon atom bonded to X2; where only one of A1 and LO is selected from carboxyl and (C1-14 alkyl)oxycarbonyl; wherein L1 is hydroxy, C1-12 alkoxy, C1-12 alkylsulfonyloxy, C6-20 arylsulfonyloxy, C7-20 arylalkyl, C6-20 aryloxy, C6-20 arylalkylcarbonyloxy, C2-8 alkylcarbonyloxy, C6-20 arylalkylcarbonylthio, C6-20 arylthio, C2-8 alkylcarbonylthio, or C1-12 alkylthio.
The invention also provides embodiments wherein Z1 is NH or NRa; X1 is O or S; Z2 is CHR1 or CHOR1, R1 being H, halogen, C1-6 alkyl, or C1-6 haloalkyl; R2 is ORf; and L0 is xe2x80x94(Cxe2x95x90X2)xe2x80x94L1, X2 being 0 and L1 being linked by a sulfur atom to the carbon atom bonded to X2. Individual compounds include lactacystin, clasto-lactacystin trifluoroethyl ester, descarboxylactacystin, lactacystin amide, and phenylacetyl lactacystin.
Embodiments of the second aspect include compounds wherein CHX4 is in the (S) configuration when X4 is hydroxyl and xe2x80x94(CH2)nxe2x80x94R3X3 is isopropyl; wherein the moiety xe2x80x94(CH2)nxe2x80x94R3X3 has between 5 and 20 carbon atoms when X4 is hydroxyl, m is O, and Z1 is NH; wherein Z1 is NH or NRa; wherein Z1 is O, S, or SO2; wherein Z1 is NRa; wherein X1 is CH2, CH(Rb), or C(Rb)(Rc); wherein Z2 is in the beta (above plane of page) configuration; wherein CHX4 is in the alpha (below plane of paper) configuration; wherein R1 is H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, or halogen; wherein R1 is a side chain of a naturally occurring xcex1-amino acid, ORd, SRd, or NRdRe; or combinations of the above.
Another embodiment of the second aspect includes compounds wherein Z1 is NH or NRa; X1 is O or S; Z2 is CHR1 in the (R) or (S) configuration, wherein R1 is H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, hydroxyl, halogen, a side chain of a naturally occurring amino acid, ORd, SRd, or NRdRe (each of Rd and Re, independently, being H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, or C2-5 alkynyl); Z3 is O or NH; X2 is O or S; and A1 is the side chain of any naturally occurring xcex1-amino acid, or is of the following formula,
xe2x80x94Yxe2x80x94(CH2)nxe2x80x94R3X3 
wherein Y is Cxe2x95x90O or CHX4 in the (R) or (S) configuration, or deleted, X4 being halogen, methyl, halomethyl, or ORh, wherein Rh is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, C1-10 acyl, C1-6 alkylsulfonyl, and C6-10 arylsulfonyl; n is 0, 1, 2, or 3; and R3 is straight chain or branched C1-8 alkylene, C3-10 cycloalkylene, phenylene, C6-14 arylalkylene, or deleted; and X3 is H, hydroxyl, thiol, carboxyl, amino, halogen, or C6-10 aryl; or R3 and X3 taken together are the side chain of any naturally occurring xcex1-amino acid.
The invention also provides a compound wherein Z1 is NH and Z3 is 0; or a compound wherein each of Z1 and Z3 is NH; or a compound wherein at least four (e.g., at least 5, or all) of the following limitations apply: Z1 is NH or NRa; X1 is O or S; Z2 is CHR1 in the (R) or (S) configuration, wherein R1 is H, C1-6 alkyl, C1-6 haloalkyl, hydroxyl, or ORd, Rd being C1-6 alkyl; Z3 is O or NH; X2 is O; Z3 is O or NH; and A1 is the side chain of any naturally occurring xcex1-amino acid, or CHX4, X4 being halogen, methyl, halomethyl, or ORh, Rh being selected from H, C1-6 alkyl, C1-6 haloalkyl, C1-10 acyl, C1-6 alkylsulfonyl, and C6-10 arylsulfonyl; R3 is straight chain or branched C1-8 alkylene, C3-10 cycloalkylene, phenylene, C6-14 arylalkylene, or deleted; m is 0, 1, or 2 and n is 0 or 1; and X3 is H.
The invention also provides a pharmaceutical composition wherein Z1 is NH or NRa; X1 is O or S; Z2 is CHR1 in the (R) or (S) configuration, wherein R1 is H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, hydroxyl, halogen, a side chain of a naturally occuring xcex1-amino acid, ORd, SRd, or NRdRe (each of Rd and Re, independently, being H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, or C2-5 alkynyl); Z3 is O or NH; X2 is O or S; and A1 is the side chain of any naturally occurring xcex1-amino acid, or is of the following formula,
xe2x80x94Yxe2x80x94(CH2)nxe2x80x94R3X3 
wherein Y is Cxe2x95x90O or CHX4 in the (R) or (S) configuration, or deleted, X4 being halogen, methyl, halomethyl, or ORh, wherein Rh is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, C1-10 acyl, C1-6 alkylsulfonyl, and C6-10 arylsulfonyl; n is 0, 1, 2, or 3; and R3 is straight chain or branched C1-8 alkylene, C3-10 cycloalkylene, phenylene, C6-14 arylalkylene, or deleted; and X3 is H, hydroxyl, thiol, carboxyl, amino, halogen, or C6-10 aryl; or R3 and X3 taken together are the side chain of any naturally occurring xcex1-amino acid.
Another embodiment includes a compound wherein X2 is O, and A1 is the side chain of any naturally occurring xcex1-amino acid, or X4 is halogen, methyl, halomethyl, or ORh, Rh being selected from H, C1-6 alkyl, C1-6 haloalkyl, C1-10 acyl, C1-6 alkylsulfonyl, and C6-10 arylsulfonyl; R3 is straight chain or branched C1-8 alkylene, C3-10 cycloalkylene, phenylene, C6-14 arylalkylene, or deleted; m is 0, 1, or 2 and n is 0 or 1; and X3 is H. Individual lactone analogs include clasto-lactacystin xcex2-lactone.
Embodiments of the third aspect include compounds wherein: when X1 is 2 singly bonded H and R1 has only one oxygen, then the fragment formula weight of R1 is at least 30 atomic mass units; when X1 is 2 singly bonded H and R1 is 2H, and A1 is alkyl, then A1 has at least 3 carbon atoms; A1, R1 and X1 taken together have at least one carbon atom, halogen, or heteratom; when X1 is two singly bonded H, and R1 is H, X2 is O, and Rh is alkyl, then Rh is C4-6 alkyl; and when n is 2, Rh is C1-6 haloalkyl.
Further embodiments of the third aspect include compounds wherein: X1 is CH2, two singly bonded H, CH(Rb) or C(Rb)(Rc); Z2 is O or S; Z2 is NH or NRj; R1 is C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, or halogen; R1 is a side chain of a naturally occuring xcex1-amino acid, ORd, SRd, or NRdRe; A1 cannot be H; R1 is in the beta configuration; X4 is in the alpha configuration; or combinations thereof.
Embodiments of the fourth aspect include compounds wherein: Z2 is O or S; Z2 is NH or NRj; X1 is O or S; R1 is C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, or halogen; R1 is a side chain of a naturally occuring xcex1-amino acid, ORd, SRd, or NRdRe;; R2 is H, C1-3 alkyl, C1-3 haloalkyl, C2-3 alkenyl, C2-3 alkynyl, C6-12 aryl, C3, heteroaryl, or halogen; R2 is H, C3-6 alkyl, C3-6 haloalkyl, C4-6 alkenyl, C4-6 alkynyl, C6-12 aryl, or C3-8 heteroaryl; A1 cannot be H; R1 is in the beta configuration; X4 is in the alpha configuration; or combinations of the above.
Further embodiments of the fifth aspect are compounds wherein: Z1 is O or S; Z2 is NH or NRj; R1 is H, C1-3 alkyl, C1-3 haloalkyl, C2-3 alkenyl, C2-3 alkynyl, or a side chain of a naturally occuring xcex1-amino acid, ORd, SRd, or NRdRe; R1 is H, C4-6 alkyl, C4-6 haloalkyl, C4-6 alkenyl, C4-6 alkynyl, halogen, or a side chain of a naturally occuring xcex1-amino acid, ORd, SRd, or NRdRe; R2 is H, C1-3 alkyl, C1-3 halo-alkyl, C2-3 alkenyl, C2-3 alkynyl, C6-12 aryl, C3-8 heteroaryl, or halogen; R2 is H, C4-6 alkyl, C4-6 haloalkyl, C4-6 alkenyl, C4-6 alkynyl, C6-12 aryl, or C3-8 heteroaryl; n Ra is C1-3 alkyl; A1 cannot be H; X4 is in the alpha configuration; or combinations thereof.
Embodiments of the sixth aspect are compounds wherein: X1 is O or S; X1 is CH2, two singly bonded H, CH(Rb), or C(Rb) (Rc); Z1 is O or S; Z1 is NH or NRa; R1 is C1-3 alkyl, C1-3 haloalkyl, C2-3 alkenyl, C2-3 alkynyl, halogen, or a side chain of any naturally occuring xcex1-amino acid; R1 is ORd, SRd, or NRdRe; R1 is C4-6 alkyl, C4-6 haloalkyl, C4-6 alkenyl, C4-6 alkynyl, a side chain of any naturally occuring xcex1-amino acid, ORd, SRd, or NRdRe; R1 and R2 taken together are a bivalent moiety; R1 is in the beta configuration; R2 is in the beta configuration; R2 is C1-3 alkyl, C1-3 haloalkyl, C6-12 aryl, C3-8 cycloalkyl, or halogen; R2 is C4-6 alkyl, C4-6 haloalkyl, C6-12 aryl, C3-8 cycloalkyl, C3-8 heteroaryl, or C3-8 heterocyclic radical; A1 has a higher fragment formula weight than A2; A1 has a lower fragment formula weight than A2; A1 is in the (R) configuration; A1 is in the (S) configuration; only one of A1 and A2 is H; A1 is a side chain of any naturally occuring xcex1-amino acid; or combinations thereof.
Embodiments of the seventh aspect are compounds wherein: X1 is O or S; X1 is CH2, two singly bonded H, CH(Rb) or C(Rb)(Rc); Z2 is O or S; Z2 is NH or NRj; R1 is C1-3 alkyl, C1-3 haloalkyl, C2-3 alkenyl, C2-3 alkynyl, hydroxyl, halogen, or a side chain of any naturally occuring xcex1-amino acid; R1 is ORd, SRd, or NRdRe; R1 is C4-6 alkyl, C4-6 haloalkyl, C4-6 alkenyl, C4-6 alkynyl, a side chain of any naturally occuring xcex1-amino acid; R1 and R2 taken together are a bivalent moiety; R2 is C1-3 alkyl, C1-3 haloalkyl, C2-3 alkenyl, azido, C2-3 alkynyl, hydroxyl, or halogen; R2 is ORf, SRf, NRfRfRg, xe2x80x94ONRfRg, xe2x80x94NRg(ORf), or xe2x80x94NRg(RSf); R2 is C4-6 alkyl, C4-6 haloalkyl, C4-6 alkenyl, azido, C4-6 alkynyl, ORf, SRf, NRfRfRg, xe2x80x94ONRfRg, xe2x80x94NRg(ORf), or xe2x80x94NRg(RSf); R1 and R2 taken together are a bivalent moiety; R1 is in the alpha configuration; R1 is in the beta configuration; R2 is in the alpha configuration; R2 is in the beta configuration; A1 is in the alpha configuration; A2 is in the beta configuration; or combinations thereof.
One embodiment of the eighth aspect is a compound where A1, A2, R12, and X1 taken together have at least one carbon atom and one heteratom. Further embodiments of the eighth aspect are compounds wherein: Z1 is NH, or NRa; X1 is O or S; X1 is CH2, or two singly bonded H; R12 is H, C1-3 alkyl, C1-3 haloalkyl, C2-3 alkenyl, or C2-3 alkynyl; R12 is C4-6 alkyl, C4-6 haloalkyl, C4-6 alkenyl, or C4-6 alkynyl; where A1, A2, R12, and X1 taken together have at least one carbon atom and one heteratom; where the fragment formula weight of A1 is greater than the fragment formula weight of A2 (e.g., by at least 30 or 60); where the fragment formula weight of A2 is greater than the fragment formula weight of A1 (e.g., by at least 15 or 50); or combinations thereof.
Embodiments of the ninth aspect are compounds wherein A1 is a side chain of any naturally occurring xcex1-amino acid; wherein A1 has a fragment formula weight of at least 50 (e.g., 70, 100, or 120).
Examples of individual compounds described herein are provided in Tables A, B, and C. 
Synthesis
The syntheses disclosed herein are organized by structure groups. A person of skill will recognize that compounds of claim 1 are related to the compounds in the compositions of claim 23, and the compounds of claim 5 are related to the compounds in the compositions of claim 30 and so on. The synthesis of the compounds in claims 1-5 rely primarily on the results reported by Uno, et al. in their enantiospecific synthesis of (+)-lactacystin from (R)-glutamate [Uno, et al., J. Am. Chem. Soc. (1994) 116:2139]. Note that in all schemes relating to claims 1-5 and 24-32, when a straight line is used to connect A1 (or anything corresponding to A1) to the rest of the molecule, a dashed line should be assumed. The straight line is used simply for clarity. All A1 are attached to the rest of the molecule on the xe2x80x9calpha,xe2x80x9d i.e., the bottom, face.) Compound A-1 (Scheme 1) serves as the starting material for all synthetic routes proposed in claim 2, for example. Thus, conversion of A-1 to A-2, in analogy to the above-mentioned work allows for introduction of R1 via standard alkylation chemistry. In the cases where R1 is hydroxyl, halide, xe2x80x94SRd, or xe2x80x94NRdRe, wherein Rd and Re each, independently, is selected from H, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, the electrophile in step b is, respectively, a N-arylsulfonyl-3-phenyloxaziridine [Davis, F. A. et al., J. Org. Chem. (1984), 49:3241], a N-halosuccinimide [Stotter, P. A. et al., J. Org. Chem. (1973) 38:2576], RdSxe2x80x94SRd or elemental sulfur [Zoretic, P. A. et al., J. Org. Chem. (1976) 41:3587] [Gassman, P. G. et al., J. Org. Chem. (1977) 42:3236], a N-arylsulfonylazide (followed by reduction of the azide to the primary amine and elaboration to NRdRe) [Evans, D. A. et al., J. Am. Chem. (1987) 109:6881]. If R1 is labile or reactive, or both, a protecting group would be introduced at this point and removed at the end of the synthesis. The preparation of compounds of type A-2 is then completed with the two-step (steps b and c) process reported by Uno [Uno, et al., (1994)].
Introduction of A1 is the next task. Uno [Uno, et al., (1994)] showed that under Lewis-acid mediated aldol addition conditions, A1 is introduced with very high facial selectivity with respect to the bicyclic aminoacid derivative A-2. This carbon-carbon bond construction allows for the A1 groups detailed in claim 2. Accordingly, conversion of A-2 to A-3, as described by Uno,
followed by treatment of A-3 with SnCl4 and an aldehyde (RfCHO, where Rf corresponds to H or to the rest of A1), in diethylether yields aldol products A-4 (or A-5 if Rf is H), following suitable protection of the hydroxyl group with a group designated here as Po. [Greene and Wuts, et al., Protective Groups in Organic Synthesis, 2nd ed., (1991)]. 
Introduction of R2 by a stereoselective, conjugate nucleophilic addition to the unsaturated carbonyl system of A-4 or A-5, followed by quenching of the resulting enolate with acidic water gives access to compounds A-6 and A-7, respectively. This process is analogous to the xe2x80x9cthree-component couplingxe2x80x9d strategy for prostaglandin synthesis of [Suzuki, et al. (1988)]. In this case, the three components are A-4 (or A-5), R2, and a proton. The diastereomers shown for A-6 and A-7 are predicted to be the major ones based on several facts. The Uno synthesis [Uno, H., et al., (1994)] used an Os04-catalyzed dihydroxylation of a compound similar to A-4 (R1=methyl, Rf=iso-propyl) to introduce a hydroxyl group corresponding to R2. This reaction proceeded with complete facial selectivity. Thus, in general, groups with appropriate reactivity (nucleophiles or electrophiles) similarly approach preferentially the xe2x80x9cbetaxe2x80x9d face (from the top) of A-4 or A-5.
Quenching of the enolate with acidic water proceeds stereoselectively. In Suzuki""s three-component coupling strategy, [Suzuki, et al., (1988)] the nucleophile and electrophile are introduced trans to each other. In our case, R2 and the proton are introduced trans to each other. In the event that compounds in which R1 and R2 are trans to each other are desired, as depicted in compounds A-12 and A-13, a similar strategy has been devised.
The basic strategy has one exception to that detailed above: The order of introduction of R1 and proton at the carbon alpha to the carbonyl is reversed. Thus, conversion of A-1 to A-9 is carried out using the same procedures as those for the preparation of A-3, omitting the alkylation step (step a). Elaboration of A-9 to A-10 and A-11 utilizes the same conditions as those which afford A-4 and A-5. Addition of R2 still occurs from the top face, but the quenching step with an electrophile corresponding to R1 now gives a trans relationship of R1 and R2, as shown in compounds A-12 and A-13.
Another advantage of these approaches to A-6, A-7, A-12, and A-13 is as follows. Should the quenching step introduce the electrophile (proton or R1) cis to R2 (opposite that predicted above), then one still has stereospecific routes to the desired compounds, except that A-4 and A-5 give A-12 and A-13, respectively, and A-10 and A-11 give A-6 and A-7.
Compounds in which A1 is H are prepared by a different strategy (Scheme 2). For example, it has been shown that when A1 is H, nucleophiles [Hanessian, et al., Synlett (1991) 10:222] and electrophiles [Griffart-Brunet, et al., Tet. Lett. (1991) 35:119] approach from the bottom face, cis to the hydrogen at Al. Thus, the key to our strategy above is that the stereoselectivity of addition is reversed when A1 is not H. The rapid preparation described below of compounds in which A1 is H takes advantage of this reversal of stereoselectivity.
Thus, bromination of A-2 by standard methods [Caine, et al., J. Org. Chem. (1985) 50:2195] gives A-14, which is utilized in conjugate addition-elimination procedures to give compounds of type A-15. Since A1 is H, catalytic hydrogenation of the olefin should install R1 and R2 in the desired cis configuration shown in compound A-16. Cleavage of the N,O-aminal may occur under such conditions, giving compound A-17. Such an occurrence is not disadvantageous since such a deprotection is the next step in the synthesis anyway. Compounds A-18 and A-19 are accessed by base-catalyzed epimerization of A-16 and A-17, respectively. 
The above strategies allow for the preparation of compounds containing any and all R1 and R2 described in claims 2 and 25, with one exception: compounds where an additional ring is fused to the lactam ring (specifically, compounds in which R1 is taken together with R2, giving a bivalent moiety which forms a C3-8 cycloalkyl, C3-8 heteroaryl, C3-8 heterocyclic radical, or C6-12 aryl). Their preparation will be discussed below (Schemes 3 and 4). Note that while only carbocyclic systems are depicted in Schemes 3 and 4, both the alicyclic and heterocyclic (aromatic and non-aromatic in both cases) classes of compounds can be prepared in this manner by choice of the appropriate reagents. Further, all substitution patterns on these rings that are normally accessible by such processes are accessible by the methods proposed below. The simplest carbocyclic variants are shown for clarity and to illustrate the general strategy to these compounds.
The synthesis of these compounds relies on the diverse chemistry of cycloaddition [Carruthers, et al., Cycloaddition Reactions in organic Synthesis (1990) and J. March, Advanced organic Chemistry, (1992) pp. 826-877] to alpha-beta unsaturated olefins. Thus, as shown in Scheme 3, [1+2] processes (e.g. diazoinsertion, cyclopropanation, epoxidation, and aziridination) with compound A-20 (see Scheme 1 for preparation of compounds of this type, e.g. A-10 and A-11) gives access to compounds of type A-21. Compounds of type A-22 are prepared with a variety of [2+2] cycloaddition processes (e.g. photocycloadditions and ketene additions). 1,3-Dipolar cycloaddition chemistry ([3+2] cycloaddtion reagents, such as nitrile oxides and azomethine ylides) prepares a wide variety compounds of the type A-23. The preparation of compounds of type A-24 is perhaps the most flexible and far-reaching of the cycloaddition chemistry 
because it relies on the Diels-Alder reaction, one of the most utilized and studied reactions in all of organic chemistry. The variety of compounds of type A-24 that are prepared by this method is far to great to detail; [Carruthers, (1990)] however, notably, bridged compounds (type A-25) are prepared by this method. [5+2] cycloadditions to give A-26 are less common, but some intriguing examples have been reported. [Wender, et al., J. Org. Chem. (1991) 56:6267 and Wender, et al., Tet. Lett. (1992) 32:6115].
While all these procedures introduce R1 and R2 cis to each other, base-catalyzed epimerization in the cases of A-24 and A-26 gives access to the trans-fused versions of these compounds, A-28 and A-29, respectively. (A-21, A-22, A-23, and A-25 can not be epimerized in this fashion, as these trans-fused rings suffer from much more ring strain than do the cis-fused systems.)
Cycloheptyl (A-26) and cyclooctyl (A-27) compounds are prepared with a slightly different process, one that is akin to that shown in Scheme 1. Fundamentally, one adds an nucleophile (abbreviated as xe2x80x9cNuxe2x80x9d) to A-20 which also has a leaving group (abbreviated as xe2x80x9cLGxe2x80x9d) attached to the other end of the incoming nucleophile, separated by the necessary number of atoms (5 in the case of A-26, 6 in the case of A-27). Thus, following the addition of the nucleophile, the resulting enolate displaces the leaving group, forming the ring. Alternatively, such a process is done stepwise in order to minimize undesired intramolecular side reactions of the nucleophile and electrophile. These and other methods for making cycloheptyl and cyclooctyl compounds from alpha-beta unsaturated carbonyl compounds have been reviewed in the chemical literature. [Petasis, N. A., et al., Tetrahedron (1992) 48:5757].
Aryl and heteroaryl compounds (e.g. compounds A-30 and A-31, Scheme 4) are prepared by [4+2] cycloaddition chemistry, followed by oxidation of the resultant cycloalkene or heterocycloalkene with dichlorodicyanoquinone (DDQ). [Pizey, N., Synth. Reagents (1977) 3:193-225]. In the case of compounds of type A-31, other types of dienes that are utilized are the hydroxyorthoxylylenes, formed by the ring opening reaction of hydroxybenzocyclobutenes. [Arnold, B. J., et al., J. Chem. Soc., (1974) 409-4153.
Thus, having prepared all the variations of R1 and R2, we turn now to the elaboration of these compounds to include all of the variations of A1 (Scheme 5).
Based on the results of [Uno, et al., (1994)] and as shown in Scheme 1, Lewis-acid catalyzed aldol additions of A-3 to RfCHO, followed by further elaboration, provide compounds of the type A-33 (Scheme 5) with the stereocenters in the configurations shown, including both configurations of R1. (A straight bond (i.e. neither bold or dashed) is shown here to represent both diastereomers at this position.) Here we will focus on the stereochemistry of the secondary hydroxyl that is included in A1, and related derivatives. While modification of conditions in the aldol addition (Scheme 1) reaction can provide the other diastereomer at this position, another method of inverting this center is to first oxidize to the ketone under standard conditions (e.g. Swern, Dess-Martin periodinane, etc.) and then reduce with the appropriate reducing agent (e.g. NaBH4, etc.) whose identity is determined by screening a number of reductants. This two-step procedure provides compounds of type A-35. Analogously, reductive amination of ketone A-34 with an amine or hydroxylamine (represented in Scheme 5 as RnNH2) and sodium cyanoborohydride (NaCNBH3) provides compounds of type 
A-36. The sense of stereochemical induction in this step can be altered by using a different reducing agent. Alternatively, by first preparing the methanesulfonate ester (xe2x80x9cmesylatexe2x80x9d) of A-33 or A-35, and then displacing the mesylate with a nucleophile (e.g. RNH2, RSxe2x80x94, ROxe2x80x94, halide, hydroxylamine, Grignard reagents, etc.) one obtains a wide variety of A1 derivatives with complete control of stereochemistry at the carbon corresponding to the secondary hydroxyl in A-33. Reduction of the mesylate (with lithium triethylborohydride (LiEt3BH)) or of a halide (with, e.g., zinc in hydrochloric acid) provides compounds of the type A-38.
Another method to produce the various A1 derivatives is depicted in Scheme 6. Compounds of the type A-39, which are prepared according to Scheme 1, are converted to those of type A-40 where Xa is a halide or trifluoromethanesulfonate ester (xe2x80x9ctriflatexe2x80x9d), for example, using standard functional group manipulation. Displacement of these leaving groups with nucleophiles gives another method for the preparation of compounds of type A-38. Subjecting iodide A-40 (Xa is I) to metal-halogen exchange conditions (e.g. activated Mg metal or tert-butyllithium) provides nucleophiles to which a variety of nucleophiles can be added, providing yet another route to compounds of type A-38.
Another strategy for the preparation of a subset of compounds of type 33 is worth noting (Scheme 7). This strategy allows for the preparation of compounds of type A-33 for all R2 discussed above and for all R1 other than hydroxyl, halide, xe2x80x94SRd,xe2x80x94NRdRe Compounds of the type A-4, A-5, A-10, and A-11 as shown in Scheme 1 (represented as compound A-41 in Scheme 7), in which R1 is attached to the lactam ring with a carbon-carbon bond, can be dihydroxylated to give compounds of type A-42, in which the hydroxyl groups have been added to the top face exclusively. [Uno, et al., (1994)] (The reasons for this stereoselectivity have been discussed above.) Following the procedure of [Uno, et al., 1994)] the tertiary hydroxyl can be removed selectively, yielding a mixture of R1 diastereomers A-43, which can be separated by a variety of chromatographic methods. Mitsunobu inversion of the secondary hydroxyl, followed by displacement of a suitably derived leaving group by any nucleophile corresponding to R2 (e.g. hydroxide, alkoxide, sulfide, Grignard reagents, amine, halide, etc.) gives compounds of the type A-44, which are a subset of compounds of the type A-33, and can therefore be elaborated into their corresponding A1 derivatives as described above.
Thus, having provided viable routes to all variations of R1, R2, and now A1 in claims 2 and 25, we now turn our attention to the installation of X1, L1, and X2 (Scheme 8).
The compound of general structure A-45 can be converted to A-46 in a two-step process involving first catalytic hydrogenolysis or mild acidic hydrolysis [Corey, E. J., et al., J. Am. Chem. Soc. (1992a), 114:10677] and then protection of the primary hydroxyl group as its tert-butyldimethylsilyl (TBS) ether under standard conditions. [Corey, E. J. and Venkateswarlu, A., J. Am. Chem. Soc. (1972), 94:6190] N-Alkylation of the amide provides access to compounds A-47. [Challis, N., The Chemistry of Amides, (1970) 734-754]. (In the cases where Rb is desired to be H, the amide can be blocked with a suitable protecting group that will be removed at the end of the synthesis. [Greene, T. W., et al., Protective Groups in Organic Synthesis (1991)].
Enamine A-48 can be prepared from A-47 under Tebbe olefination conditions. [Pine, S. H., et al., J. Org. Chem. (1985) 50:1212]. Thioamide A-49 can be prepared bytreatment of A-47 with bis(tricyclohexyltin)sulfide and 
boron trichloride. [Steliou, K., et al., J. Am. Chem. Soc., (1982) 104:3104]. Substituted enamines A-50 can be prepared by addition of the appropriate Grignard reagent or alkyllithium to A-47 or A-49, followed by acidic hydrolysis, and separation of the E and Z olefin isomers. [Aguerro, A., et al., J. Chem. Soc., Chem. Commun. (1986) 531 and Schrock, R. R., J. Am. Chem. Soc., (1976) 98:5399, and Hansen, C., J. Org. Chem. (1973) 38:3074]. Pyrrolidines of type 51 can be prepared by reduction of A-47 with lithium aluminum hydride [March, J., Advanced Organic Chemistry (1992) 826-877 and Gaylord, J., Reduction With Complex Metal Hydrides (1956)] 544-636] or alternatively by reduction of A-49 with Raney nickel. [Belen""kii, W., Chemistry of Organosulfur Compounds 91990) 193-228].
Taken together, these procedures constitute a synthesis of compounds of the general structure A-52, which are converted to A-53 following, for example, the procedure of Uno, et al. [Uno, H., et al., (1994)] (Scheme 9). Compounds A-53 are converted to analogues A-54a via oxidation to the acid and coupling with L1 (all of which can be prepared by standard methods) under the conditions utilized by Corey and Reichard [Corey, E. J., et al., (1992a]. Sulfurization of these compounds with Lawesson""s reagent [Cava, M. P., et al., Tetrahedron (1985) 41:5061] gives thiono analogues A-55a. Compounds A-54b are prepared via addition of a nucleophile (e.g. CF3) [Francese, C., et al., J. Chem. Soc., Chem. Commun (1987) 642], corresponding to L2 to the aldehydes A-54b followed by oxidation of the alcohol with Dess-Martin periodinane. [Linderman, R. J., et al., Tet. Lett. (1987) 28:42593. Sulfurization of these compounds with Lawesson""s reagent [Cava, M. P., et al., (1985)] gives thiono analogues A-55b. Epoxides of the type A-55c are prepared from A-53 or A-54b by the method of Johnson 
[Johnson, C. R., Acc. Chem. Res. (1973) 6:341], thuscompleting the synthesis of all the analogues detailed in this section.
The preparation of the compounds covered by claims 4 and 31 relies primarily on the methods of Seebach and of Corey. Thus, as shown in Scheme 10, epoxides of the type A-58 are prepared according to the procedure of Corey, et al., [Corey, E. J., et al., (1992b)] from 61 via compound 62. R2 is thus installed by choosing the appropriate aldehyde for the aldol reaction in step a. Corey [Corey, E. J., et al., (1992b)] showed that epoxides A-58 are opened stereospecifically to give A-59. Thus, Z1 is installed by treating A-58 with a nucleophile corresponding to Z1. (Pz refers to a protecting group for Z1.) Installation of Z2 is accomplished by conversion of the hydroxyl A-59 to a leaving group, for example, a tosylate, and displacement of the leaving group with a nucleophile corresponding to Z2. (Pz, refers to a protecting group for Z2).
The results of Seebach [Seebach, D., et al., Helv. Chim. Acta. (1987) 70:1194] are used in the next part of the synthesis. Conversion of A-60 to A-61 allows for enolization and alkylation with an electrophile corresponding to A1, yielding A-62. Seebach showed that such alkylations give predominantly the diastereomer shown.
After reduction of the ester and protection of the resulting primary alcohol as the TBS ether, removal of the tert-butylmethylene protecting group, enables conversion to compounds of either the type A-63 (by treatment with, for example, formaldehyde and an acid catalyst) or the type A-64 (by treatment with, for example, CDI) is effected.
Compound is a key intermediate in the completion of thesynthesis of the compounds in claims 4 and 31, (Scheme 11). Compounds of the type A-65 are prepared from A-64 under Tebbe olefination conditions. [Pine, S. H., et al., (1985)]. Compounds of the type A-66 are prepared by treatment of A-64 with bis(tricyclohexyltin)sulfide and boron trichloride. [Steliou, K., et al., (1982)]. Compounds of the type A-67 are prepared by addition of the appropriate Grigard reagent or alkyllithium to A-64 or A-66, followed by acidic hydrolysis, and separation of the E and Z olefin isomers. [Aguerro, A., et al., (1986) and Schrock, R. R., (1976), and Hansen, C., et al., (1973)]. Compounds of the type A-63, prepared in Scheme 10, are also prepared by reduction of A-64 with lithium aluminum hydride [March, J., (1992), and Gaylord, J. (1956)] or alternatively by reduction of A-66 with Raney nickel. [Belen""kii, W., (1990)].
Taken together, compounds of the type A-63, A-64, A-65, A-66, and A-67 are of the general class A-68, which are converted the general class of lactacystin analogues A-69 by fluoride deprotection of the TBS ether, oxidation of the resulting primary alcohol to the carboxylic acid, via the aldehyde analogues, in, for example, the two-step process shown, and coupling with Li using the method of Corey and Reichard, [Corey, E. J., et al., (1992a)] and removal of the protecting groups on Z1 and Z2 (if necessary). Lactacystin analogues A-71 are prepared by treating A-69 with Lawesson""s reagent [Cava, M. P., et al., (1985)]. Analogues A-70 are also prepared from A-68 by fluoride deprotection of the TBS ether, oxidation of the resulting primary alcohol to the aldehyde, addition of nucleophile (e.g., CF3) [Francese, C. et al. 1987, supra] Dess-Martin periodinane oxidation, sulfurization with Lawesson""s reagent (if desired) [Cava, M. P., 91985)], and deprotection of Z2 and Z2, if necessary. Epoxides of the type A-72 are also prepared from A-68 by fluoride deprotection of the TBS ether, oxidation of the 
resulting primary alcohol to the aldehyde, addition of a nucleophile corresponding to L2, Dess-Martin periodinane oxidation, addition of dimethyloxosulfonium methylide, and deprotection of Z1 and Z2 (if necessary). Thus, the above strategy prepares all the compounds in claims 4 and 31.
The preparation of the compounds covered by claim 1 relies primarily on the methods of Evans and of Corey. Thus, as shown in Scheme 12, chiral oxazolidinones of the type A-73 are enolized and alkylated with benzyloxy-bromomethane, thereby installing R1. [Evans, D. A., et al. J. Am. Chem. Soc., (1982) 104:1737]. (Only one configuration of R1 is obtained in the reaction. The other configuration of R1 is obtained by using the opposite enantiomer of the chiral oxazolidinone.) Conversion of A-74 to aldehydes of the type A-75, and chiral, boron-mediated (using A-76) aldol condensation with tert-butylbromoacetate gives [Corey, E. J., et al., (1992b)] a secondary alcohol that is protected as the tert-butyldimethylsilyl (TBS) ether to give compounds of the type A-77. Displacement of the bromide with a nucleophile [Corey, E. J., et al. (1992b)] corresponding to Z1 yields A-78, which can be converted to compounds of the type A-79. As discussed above regarding claim 4, such compounds can be enolized and alkylated stereospecifically with electrophiles corresponding to A1, giving A-80. Standard functional group manipulation yields tosylates of the type A-81. Displacement of the tosylate with a nucleophile corresponding to R2, [Hanessian, S., et al., J. Org. Chem. (1989) 54:5831] gives compounds of the type A-82, the primary alcohol of which is deprotected via, for example, catalytic hydrogenolysis, and cyclized to compounds A-83 after oxidation of the primary alcohol to the carboxylic acid. 
As shown in Scheme 13, compounds of the type A-84, in which R1 is in either the (R) or (S) configuration, are converted into a variety of X1 variants. Compounds of the type A-85 are prepared from A-84 under Tebbe olefination conditions. [Pine, et al., (1985)] Compounds of the type A-86 are prepared by treatment of A-84 with bis(tricyclohexyltin)sulfide and boron trichloride. [Stelious, K., et al., (1982)]. Compounds of the type A-87 are prepared by addition of the appropriate Grignard reagent or alkyllithium to A-84 or A-86 followed by acidic hydrolysis, and separation of the E and Z olefin isomers. [Aguerro, A., et al., (1986), Schrock, R. R., (1976) and Hansen C., et al., (1973)]. Compounds of the type A-88 are prepared by reduction of A-84 with lithium aluminum hydride [March, J. (1992), and Gaylord, J., (1956)] or alternatively by reduction of A-86 with Raney nickel. [Belen""kii, W., (1990)]. When Z1 is S in A-88, oxidation to the cyclic sulfone variants is effected with, for example, KHSO5. [Trost, B. M., et al., Tet. Lett. (1981) 22:1287].
Taken together, compounds of the type A-84, A-85, A-86, A-87, A-88, and A-89 are of the general class A-90, which are converted analogues A-91, following for example, the procedure of Uno, et al. [Uno, H., et al., (1994)] (Scheme 13b). Compounds A-91 are converted to analogues A-92 via oxidation to the acid and coupling with L1 (all of which can be prepared by standard methods) under the conditions utilized by Corey and Reichard. [Corey, E. J., et al., (1992a)]. Sulfurization of these compounds with Lawesson""s reagent gives thiono analogues A-93. Compounds A-94 are prepared via addition of a nucleophile (e.g. CF3) [Francese, C., et al., (1987)] corresponding to L2 to the aldehydes A-91 followed by oxidation of the alcohol with Dess-Martin periodinane. Sulfurization of these compounds 
with Lawesson""s reagent gives thiono analogues A-95. Epoxides of the type A-96 are prepared from A-91 or A-94 by the method of Johnson, thus completing the synthesis of all the analogues detailed in this section. Note: As above in all Schemes relating to claim 6, when a straight line is used to connect A2 (or anything corresponding to A2) to the rest of the molecule, a dashed line should be assumed. The straight line is used simply for clarity. All A2 are attached to the rest of the molecule on the xe2x80x9calphaxe2x80x9d face (i.e. the bottom face).
The proposed syntheses of all of the compounds listed in claim 17 rely on a key intermediate from Scheme 9. As shown in Scheme 14, compounds of type B-1 are analogous to a subset of compounds A-53, whose various preparations were described in Schemes 1, 2, and 5-8, and described in the accompanying text.
In order to prepare analog B-2, the method of Corey, et al. is utilized. [Corey, E. J., et al., Tet. Lett. (1993) 34:6977]. Sulfurization of B-2 with, for example, Lawesson""s reagent [Cava, M. P., et al., (1985)] provides analogs B-3.
The preparation of compounds of type B-1 is summarized in Scheme 15. Thus the same starting material (A-1) as that used in Scheme 1 is elaborated to all of the compounds B-2 by the same methods as those used in Schemes 1, 2, 5, 6, and 8. There are then two possible general strategies for the conversion of these intermediates to B-2. The first is analogous to that detailed in Schemes 1, 2, 5, 6, 8, and 9, in which Z5 (suitably activated or protected) serves as the nucleophile in the relevant reactions.
The other strategy, based on that shown in Scheme 7 is also depicted in Scheme 15. Thus, dihydroxylation of B-4 gives B-5, which is then be deoxygenated as before, giving B-6. Separation of the diastereomers, Mitsunobu 
inversion of the secondary hydroxyl, and displacement of a leaving group derived from the Mitsunobu product provides compounds of the type B-7, which are then elaborated to B-1 as described in Schemes 8 and 9.
The proposed syntheses of all of the compounds listed in claim 17 rely on intermediate A-81 from Scheme 12. As shown in Scheme 16, compounds of type B-li are analogous to a subset of compounds A-81, whose various preparations are illustrated in Scheme 12, and described in the accompanying text. Conversion of compounds of the type B-11 to B-12 is performed in analogy to Scheme 12. Compounds of the type B-13 are prepared from B-12 following the relevant steps in Schemes 13a and 13b.
In order to prepare analogs B-14, the method of Corey, et al. is utilized. [Corey, E. J., et al., (1993)] Sulfurization of B-14 with, for example, Lawesson""s reagent (Cava, M. P., et al., (1985)] provides analogs B-15.
The preparation of the compounds covered by claim 7 relies on a key intermediate from Scheme 7. As shown in Scheme 17, compound C-1 is analogous to A-41, whose preparation is illustrated in Scheme 7 and described in the accompanying text. Following the work of Fuchs, [Hutchinson, D. K., et al., J. Am. Chem. Soc. (1987)] C-1 is treated sequentially with bis(benzyloxymethyl)lithium cuprate and acidic water, giving compounds of the type C-2. Both configurations of R7 can be accessed. See Schemes 1 and 2 for details. As in Scheme 8, mild acidic hydrolysis, protection of the primary hydroxyl, and alkylation or protection of the nitrogen gives compounds C-2.
Deprotection of the benzyl group by, for example, catalytic hydrogenolysis followed by displacement of the derived tosylate with a nucleophile corresponding to Z7, accesses compounds C-3, in which Z7 is protected, if 
necessary. Elaboration to carboxylic acids C-4 follows the same set of reactions in Schemes 8 and 9, allowing the preparation of all X9 variants. Deprotection of Z7, if necessary, followed by cyclization with, for example, DCC, yields bicyclic structures C-5 which if desired are sulfurized with Lawesson""s reagent [Cava, M. P., et al., (1985)] to give C-6.
The preparation of the compounds covered by the right-hand structure in claim 7 relies on a key intermediate from Scheme 9. As shown in Scheme 18, compound C-11 is analogous to a subset, defined by Z7, of the class of compounds A-52, whose preparation is illustrated in Schemes 1-8, and described in the accompanying text. Deprotection of the primary hydroxyl with, for example, TBAF in THF and oxidation via, for example, under Swern oxidation conditions to the aldehydes C-12.Homologation of the aldehyde with, for example, the phosphorous ylide derived from triphenylmethoxymethylphosphonium chloride [Jamison, T. F., et al., J. Am. Chem. Soc., (1994) 116:5505] yields enolethers of the type C-13, which are converted to carboxylic acids C-14 with acidic hydrolysis and oxidation of the resulting aldehyde with, for example, buffered NaClO2 oxidation. [Corey, E. J., et al., (1992a)].
Deprotection of Z7, if necessary, followed by cyclization with, for example, DCC, [Klausner, Y. S. et al., Synthesis (1972) 453] yields bicyclic structures C-15 which if desired are sulfurized with Lawesson""s reagent [Cava, M. P., (1985)] to give C-16.
The preparation of the compounds covered by claims 8 and 39 relies primarily on the boron-enolate mediated asymmetric aldol procedures of Evans [Evans, D. A., et al., J. Am. Chem. Soc., (1981) 103:2127] and on the results of studies on intramolecular radical 
cyclizations. [Curran, D. P., Comprehensive organic Synthesis, (1991) 4:779-831].
Esters C-21 (Scheme 19), prepared by standard methods, are deprotonated and then alkylated with benzyloxybromo-methane and reduced with DIBAL-H to give aldehydes C-22 as a racemic mixture. Addition of C-22 to an boron enolate derived from chiral oxazolidinone imides C-23 [Evans, D. A., et al., (1981)] yields C-24 as a mixture of A3 diastereomers. The configurational identity of the other two new stereocenters is established in this reaction and is not affected by the #3 configuration of A3.
Conversion to aldehydes C-25 in a six-step procedure (for example, tosylation of the secondary hydroxyl, displacement with a nucleophile corresponding to Z7, catalytic hydrogenolysis of the benzyl group, oxidation of the alcohol to the carboxylic acid and esterification) allows for preparation of the substrate for the intramolecular radical cycloaddition. Thus, treatment of the aldehyde with the Gilbert reagent [Gilbert, J. C., et al., J. Org. Chem. (1982) 47:1837] and replacement of the enolizable hydrogen with iodine gives an acetylenes C-26, which, after deprotection of Z7, if necessary, and cyclization with, for example, BOP-Cl, affords C-27.
Exposure of C-27 to atom-transfer, intramolecular radical cyclization conditions [Curran, D. P., et al., Tet. Lett. (1987) 28:2477 and Curran, D. P., et al., J. Org. Chem. (1989) 54:3140] gives compounds C-28. This reaction deserves further comment. The stereochemistry of A1 and the iodine atom are inconsequential because the radical generated from the iodine can interconvert under the reaction conditions. Further, only one of the radical diastereomers will be able to cyclizexe2x80x94that giving the cis-4,5 ring system depicted, because the trans-4,5 suffers from much greater ring strain. 
This cyclization is favored for two other reasons. First, it is a 5-endo-dig type cyclization and therefore favored according to Baldwin""s rules for cyclization. [Baldwin, J. E., J. Chem. Soc., Chem. Commun. (1976) 734]. Second, the atom transfer conditions used are ideal because the resulting vinylic radical is very reactive and is rapidly quenched by the iodine radical. [Curran, D. P., et al., J. Am. Chem. Soc., (1986) 108:2489].
Elaboration of C-28 to C-29 is accomplished using, for example, standard Stille [Stille, J. K., Angew. Chem., Int. Ed. Engl. (1986) 25:504] or Heck [Heck, R. F., Comprehensive Organic Synthesis, (1991) 4:833-863] type coupling conditions using a suitable metallic derivative (M is, e.g., tributylstannyl) of R9. Sulfurization with Lawesson""s reagent [Cava, M. P., et al., (1985)] gives compounds C-30, thus completing the preparation of all the compounds claimed in claims 8 and 39.
The preparation of the compounds covered by claims 9 and 40 relies primarily on intermediate B-2, whose preparation is described in Schemes 13a, 13b and 14 and discussed in the accompanying text. As shown in Scheme 20, C-31 is deprotonated with a suitable base, and the resulting enolate is treated with, for example, the triflate source PhN(Tf)2 [McMurry, J. E., et al., Tet. Lett. (1983) 24:979]. The vinyl triflates C-32 are treated with, for example, a catalytic amount of (Ph3P)4Pd and a suitable metallic derivative [Stille, J. K. (1986) and Heck, R. F. (1991)] (M is, e.g., tributylstannyl) of R9, giving analogues C-33, which can be treated with Lawesson""s reagent [Cava, M. P., et al., (1985)] gives compounds C-34, thus completing the preparation of all the compounds in this section.
The preparation of the compounds covered by claims 10 and 41 relies primarily on intermediate A-38, whose 
preparation is illustrated in Schemes 1, 5, and 6, anddiscussed in the accompanying text. Compounds of the type C-41 (Scheme 21) can therefore be prepared with either the (R) or (S) configuration of Z7 by following the procedures illustrated in Schemes 1-6, and discussed in the accompanying text.
Mild acidic hydrolysis of C-41, [Corey, E. J., et al., (1992a)] followed by protection of the primary hydroxyl [Corey, E. J., et al., (1972)] allows for installation of A4 via standard alkylation procedures, [Challis, N. (1970)] giving C-42. Elaboration of these intermediates to C-43 is performed as described for the analogous compounds in Scheme 8. Deprotection of the primary hydroxyl allows for oxidation to the carboxylic acids C-44, which are cyclized with, for example, BOP-Cl, giving analogues C-45, which can be treated with Lawesson""s reagent [Cava, M. P., et al., (1985)] gives compounds C-46, thus completing the preparation of all the compounds in claims 10 and 41.
The preparation of the compounds covered by claims 11 and 42 follows the strategy illustrated in Schemes 1, 3, 4, 5, 8, and 9, and described in the accompanying text. Thus, as shown in Scheme 22, C-51, being analogous to A-2 is elaborated to C-52 following the same methods used in Scheme 1 using an aldehyde containing A5 and a protected form of Z7 in either the (R) or (S) configuration. Protection of the secondary hydroxyl gives compounds C-52, [Corey, E. J., et al., (1972)] which can be treated with a nucleophile corresponding to R9 and subsequent quenching with acidic water, giving C-53. The reasons governing the indicated stereoselectivity of the nucleophilic addition and aqueous quenching have been discussed in the text accompanying Scheme 1.
Deprotection and subsequent conversion of the secondary hydroxyl to the tosylate allows for 
introduction of A3 via displacement of the tosylate with a nucleophile corresponding to A3 to give compounds of the type C-54. [Hanessian, S., (1980)]. Elaboration of these compounds to C-55 is performed with the methods illustrated in Scheme 8 and described in the accompanying text. Deprotection of the primary hydroxyl allows for oxidation to the carboxylic acids C-56, which are cyclized with, for example, DCC, [Klausner, Y. S., et al., (1972)] following deprotection of Z7, if necessary, to give analogues C-57. Sulfurization with Lawesson""s reagent [Cava, M. P., (1985)] gives compounds C-58, thus completing the preparation of all the compounds in claims 12 and 42.
The preparation of the compounds covered by claims 13 and 43 relies primarily on the results of Lubell, et al., J. Org. Chem., 1990 55:3511. Thus, as shown in Scheme 23, L-serine (C-61) is converted to ketones C-62 by known procedures. (PhFl is an abbreviation of 9-phenyl-9-fluorenyl.) Standard Wadsworth-Emmons olefination of C-62 yields either C-63 or C-64, or a mixture thereof. The composition of the product mixture is of no consequence, since both olefin isomers yield the same diastereomer in the next reaction.
Thus, a cuprate reagent derived from A1 approaches from the back face of both C-63 or C-64 since the very bulky PhFl group blocks the other face completely, giving C-65 as the major diastereomer. C-65 is converted to C-66 in a five-step procedure (reduction of the methyl ester to the aldehyde and protection as the dimethyl acetal, deprotection of the oxazolidinone, and oxidation of the primary alcohol to the carboxylic acid.)
As depicted in Scheme 24, DCC-mediated coupling, for example, of C-66 with an amine, yields amides C-67. Taken together, C-66 and C-67 comprise the general class of compounds C-88. The PhFl group is removed by 
catalytic hydrogenolysis, and the free amine alkylated with an electrophile corresponding to R12 (reductive amination with an aldehyde or ketone and NaCNBH3 is another option) to give compounds C-69. Acidic hydrolysis of the dimethyl acetal yields intermediate aldehyde C-70, which cyclizes to analogues C-73.
Tebbe olefination of C-71 provides compounds C-73. Treatment of C-71 with Lawesson""s reagent provides compounds C-72, which can be desulfurized with Raney Nickel to give compounds C-74, thus completing the synthesis of all the compounds in claims 13 and 43.
The preparation of the compounds covered by claims 14 and 44 relies primarily on the results of Dener, et al., [J. Org. Chem., 1993, 58:1159], and on those of Evans et al.,[J. Am. Chem. Soc., 1981, 103:2127]. Thus, as shown in Scheme 25, D-aspartic acid (C-81) is protected and alkylated with an electrophile corresponding to A6 to give compounds C-82, with the major diastereomer to be that shown. Dener et al.,[J. Org. Chem., 1993, 58:1159]. (PhFl is an abbreviation of 9-phenyl-9-fluorenyl.) Site-specific DIBAL-H reduction (owing to the extreme size of the PhFl group) affords aldehyde C-83.
A diastereoselective boron-mediated aldol addition is the next step. Following the procedure of Evans, et al., J. Am. Chem. Soc., (1981) 103:2127, one obtains C-85, after TBS protection of the secondary hydroxyl. The oxazolidinone is removed with lithium hydroperoxide, the PhFl group removed with catalytic hydrogenolysis, and lactam formation accomplished under, for example, DCC conditions, to give C-86. Removal of the TBS group with TBAF and lactone formation (saponification of the methyl ester with LiOH, followed by DCC coupling may be necessary) yields C-87, which is elaborated to C-88 by alkylating the amide nitrogen with a nucleophile corresponding to Rd. Epimerization with DBU provides access to compounds C-89. Treatment of C-89 with Lawesson""s reagent, Cava et al., Tetrahedron, 1985, 41: 5061, yields compounds C-90, thus completing the preparation of all compounds in this section. Examples 13-21 below are individual syntheses. 
Use
The disclosed compounds are used to treat conditions mediated directly by the proteolytic function of the proteasome such as muscle wasting, or mediated indirectly via proteins which are processed by the proteasome such as NF-xcexaB. The proteasome participates in the rapid elimination and post-translational processing of proteins (e.g., enzymes) involved in cellular regulation (e.g., cell cycle, gene transcription, and metabolic pathways), intercellular communication, and the immune response (e.g., antigen presentation). Specific examples discussed below include xcex2-amyloid protein and regulatory proteins such as cyclins and transcription factor NF-xcexaB. Treating as used herein includes reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in manner to improve or stabilize the subject""s condition.
Alzheimer""s disease is characterized by extracellular deposits of xcex2-amyloid protein (xcex2-AP) in senile plaques and cerebral vessels. xcex2-AP is a peptide fragment of 39 to 42 amino acids derived from an amyloid protein precursor (APP). At least three isoforms of APP are known (695, 751, and 770 amino acids). Alternative splicing of mRNA generates the isoforms; normal processing affects a portion of the xcex2-AP sequence, thereby preventing the generation of xcex2-AP. It is believed that abnormal protein processing by the proteasome contributes to the abundance of xcex2-AP in the Alzheimer brain. The APP-processing enzyme in rats contains about ten different subunits (22 kDa-32 kDa). The 25 kDa subunit has an N-terminal sequence of X-Gln-Asn-Pro-Met-X-Thr-Gly-Thr-Ser, which is identical to the xcex2-subunit of human macropain. Kojima, S. et al., Fed. Eur. Biochem. Soc., (1992) 304:57-60. The APP-processing enzyme cleaves at the Gln15xe2x80x94Lys16 bond; in the presence of calcium ion, the enzyme also cleaves at the Metxe2x88x921-Asp1 bond, and the Asp1-Ala2 bonds to release the extracellular domain of xcex2-AP.
One embodiment, therefore, is a method of treating Alzheimer""s disease, including administering to a subject an effective amount of a compound (e.g., pharmaceutical composition) having a formula disclosed herein. Such treatment includes reducing the rate of xcex2-AP processing, reducing the rate of xcex2-AP plaque formation, and reducing the rate of xcex2-AP generation, and reducing the clinical signs of Alzheimer""s disease.
Other embodiments of the invention relate to cachexia and muscle-wasting diseases. The proteasome degrades many proteins in maturing reticulocytes and growing fibroblasts. In cells deprived of insulin or serum, the rate of proteolysis nearly doubles. Inhibiting the proteasome reduces proteolysis, thereby reducing both muscle protein loss and the nitrogenous load on kidneys or liver. Proteasome inhibitors are useful for treating conditions such as cancer, chronic infectious diseases, fever, muscle disuse (atrophy) and denervation, nerve injury, fasting, renal failure associated with acidosis, and hepatic failure. See, e.g., Goldberg, U.S. Pat. No. 5,340,736 (1994). Embodiments of the invention therefore encompass methods for: reducing the rate of muscle protein degradation in a cell, reducing the rate of intracellular protein degradation, reducing the rate of degradation of p53 protein in a cell, and inhibiting the growth of p53xe2x80x94related cancers). Each of these methods includes the step of contacting a cell (in vivo or in vitro, e.g., a muscle in a subject) with an effective amount of a compound (e.g., pharmaceutical composition) of a formula disclosed herein.
Another protein processed by the proteasome is NF-xcexaB, a member of the Rel protein family. The Rel family of transcriptional activator proteins can be divided into two groups. The first group requires proteolytic processing, and includes p50 (NF-xcexaB1, 105 kDa) and p52 (NF-xcexa2, 100 kDa). The second group does not require proteolytic processing, and includes p65 (RelA, Rel (c-Rel), and RelB). Both homo- and heterodimers can be formed by Rel family members; NF-xcexaB, for example, is a p50-p65 heterodimer. After phosphorylation and ubiquitination of IxcexaB and p105, the two proteins are degraded and processed, respectively, to produce active NF-xcexaB which translocates from the cytoplasm to the nucleus. Ubiquitinated p105 is also processed by purified proteasomes. Palombella et al., Cell (1994) 78:773-785. Active NF-xcexaB forms a stereospecific enhancer complex with other transcriptional activators and, e.g., HMG I(Y), inducing selective expression of a particular gene.
NF-xcexaB regulates genes involved in the immune and inflammatory response, and mitotic events. For example, NF-xcexaB is required for the expression of the immunoglobulin light chain xcexa gene, the IL-2 receptor xcex1-chain gene, the class I major histocompatibility complex gene, and a number of cytokine genes encoding, for example, IL-2, IL-6, granulocyte colony-stimulating factor, and IFN-xcex2. Palombella et al., (1994). Some embodiments of the invention include methods of affecting the level of expression of IL-2, MHC-I, IL-6, IFN-xcex2 or any of the other previously-mentioned proteins, each method including administering to a subject an effective amount of a compound of a formula disclosed herein.
NF-xcexaB also participates in the expression of the cell adhesion genes that encode E-selectin, P-selectin, ICAm, and VCAM-1, Collins, T., Lab. Invest. (1993) 68:499-508. One embodiment of the invention is a method for inhibiting cell adhesion (e.g., cell adhesion mediated by E-selectin, P-selectin, ICAm, or VCAM-1), including contacting a cell with (or administering to a subject) an effective amount of a compound (e.g., pharmaceutical composition) having a formula disclosed herein.
NF-xcexaB also binds specifically to the HIV-enhancer/promoter. When compared to the Nef of mac239, the HIV regulatory protein Nef of pbj14 differs by two amino acids in the region which controls protein kinase binding. It is believed that the protein kinase signals the phosphorylation of I-xcexaB, triggering IxcexaB degradation through the ubiquitin-proteasome pathway. After degradation, NF-xcexaB is released into the nucleus, thus enhancing the transcription of HIV. Cohen, J., Science, (1995) 267:960. Two embodiments of the invention are a method for inhibiting or reducing HIV infection in a subject, and a method for decreasing the level of viral gene expression, each method including administering to the subject an effective amount of a compound of a formula disclosed herein.
Complexes including p50 are rapid mediators of acute inflammatory and immune responses. Thanos, D. and Maniatis, T., Cell (1995) 80:529-532. Intracellular proteolysis generates small peptides for presentation to T-lymphocytes to induce MHC class I-mediated immune responses. The immune system screens for autologous cells that are virally infected or have undergone oncogenic transformation. Two embodiments of the invention are a method for inhibiting antigen presentation in a cell, including exposing the cell to a compound of a formula described herein, and a method for suppressing the immune system of a subject (e.g., inhibiting transplant rejection), including administering to the subject an effective amount of a compound of a formula described herein.
In addition, the invention provides a method for treating inflammation, wherein the method includes administering to a subject an effective anti-inflammatory amount of a pharmaceutical composition containing a compound of a formula described herein. Inflammation can be a primary or secondary responses associated with (a) injury such as a cut, laceration, puncture wound, (b) infection (including infected surgical incisions) by one or more viruses, bacteria, mycobacteria, microorganisms, parasites, and fungi, (c) allergies, (d) a disease state, (e) surgery (e.g., transplantation), or (f) a combination thereof.
Allergies are primary inflammatory responses to antigens or allergens. Sources of allergens include plants (e.g., grass or tree pollen), animals (e.g., dander, venom, urine, execreta from dogs, cats, insects, and snakes), and fungi. In addition to allergens such as rye grass, ragweed, and Japanese cedar pollen, certain foods or food components (e.g., eggs, milk, shellfish, strawberries, chocolate), vaccines, and drugs (e.g., penicillin) can induce allergic reactions in certain individuals.
Disease states include rheumatoid arthritis, scleroderma, rheumatic fever, inflammatory bowel disease (e.g., Crohn""s disease and ulcerative colitis), diabetes mellitus, myasthenia gravis, multiple sclerosis, Guillain-Barre syndrome, conjuctiva of the eye, systemic lupus erythematosus, encephalitis, Adult Respiratory Distress Syndrome, psoriasis, emphysema, Alzheimer""s disease, and muscular dystrophy.
The invention provides a method of treating inflammation induced by organ or tissue transplantation. This method includes administering to a patient who has undergone or is about to undergo transplantation a composition containing a compound having a formula disclosed herein. Transplantations include bone marrow, solid organ (e.g., kidney, lungs, heart, pancreas, liver, and skin), or tissues.
Certain proteasome inhibitors block both degradation and processing of ubquitinated NF-xcexaB in vitro and in vivo. Proteasome inhibitors also block IxcexaB-xcex1 degradation and NF-xcexaB activation, Palombella, et al.; and Traenckner, et al., EMBO J. (1994) 13:5433-5441. One embodiment of the invention is a method for inhibiting IxcexaB-xcex1 degradation, including contacting the cell with a compound of a formula described herein. A further embodiment is a method for reducing the cellular content of NF-xcexaB in a cell, muscle, organ, or subject, including contacting the cell, muscle, organ, or subject with a compound of a formula described herein.
Other eukaryotic transcription factors that require proteolytic processing include the general transcription factor TFIIA, herpes simplex virus VP16 accessory protein (host cell factor), virus-inducible IFN regulatory factor 2 protein, and the membrane-bound sterol regulatory element-binding protein 1.
Other embodiments of the invention are methods for affecting cyclin-dependent eukaryotic cell cycles, including exposing a cell (in vitro or in vivo) to a compound of a formula disclosed herein. Cyclins are proteins involved in cell cycle control. The proteasome participates in the degradation of cyclins. Examples of cyclins include mitotic cyclins, G1 cyclins, (cyclin B). Degradation of cyclins enables a cell to exit one cell cycle stage (e.g., mitosis) and enter another (e.g., division). It is believed all cyclins are associated with p34cdc2 protein kinase or related kinases. The proteolysis targeting signal is localized to amino acids 42-RAALGNISEN-50 (destruction box). There is evidence that cyclin is converted to a form vulnerable to a ubiquitin ligase or that a cyclin-specific ligase is activated during mitosis. Ciechanover, A., Cell, (1994) 79:13-21. Inhibition of the proteasome inhibits cyclin degradation, and therefore inhibits cell proliferation (e.g., cyclin-related cancers). Kumatori et al., Proc. Natl. Acad. Sci. USA (1990) 87:7071-7075. One embodiment of the invention is a method for treating a proliferative disease in a subject (e.g., cancer, psoriasis, or restenosis), including administering to the subject an effective amount of a compound of a formula disclosed herein. Chronic or acute inflammation can result from transplantation rejection, arthritis, rheumatoid arthritis, infection, dermatosis, inflammatory bowel disease, asthma, osteoporosis, and autoimmune diseases. Rejection or inflammation can occur in transplanted tissues or organs of any type, including heart, lung, kidney, liver, skin grafts, and tissue grafts. The invention also encompasses a method for treating cyclin-related inflammation in a subject, including adminstering to a subject an effective amount of a compound of a formula described herein.
Additional embodiments are methods for affecting the proteasome-dependent regulation of oncoproteins and methods of treating or inhibiting cancer growth, each method including exposing a cell (in vivo, e.g., in a subject or in vitro) to a compound of a formula disclosed herein. HPV-16 and HPV-18-derived E6 proteins stimulate ATP- and ubiquitin-dependent conjugation and degradation of p53 in crude reticulocyte lysates. The recessive oncogene p53 has been shown to accumulate at the nonpermissive temperature in a cell line with a mutated thermolabile E1. Elevated levels of p53 may lead to apoptosis. Examples of proto-oncoproteins degraded by the ubiquitin system include c-Mos, c-Fos, and c-Jun. One embodiment is a method for treating p53-related apoptosis, including administering to a subject an effective amount of a compound of a formula disclosed herein.
Treatment of cancer prevents, alleviates, or ameliorates one or more primary or secondary phenomena associated with the initiation, progression, and metastasis of tumors, especially malignant tumors, e.g., a growth of tissue wherein cell multiplication is uncontrolled. Malignant tumors show a greater degree of anaplasia than do benign tumors. The invention provides a method of treating cancer including administering to a subject an effective anti-cancer amount of a pharmaceutical composition described herein, wherein the cancer is selected from carcinoma, lymphoma, sarcoma, and myeloma.
Examples of carcinomas include adenocarcinoma, acinic cell adenocarcinoma, adrenal cortical carcinomas, alveoli cell carcinoma, anaplastic carcinoma, basaloid carcinoma, basal cell carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, renaladinol carcinoma, embryonal carcinoma, anometroid carcinoma, fibrolamolar liver cell carcinoma, follicular carcinomas, giant cell carcinomas, hepatocellular carcinoma, intraepidermal carcinoma, intraepithelial carcinoma, leptomanigio carcinoma, medullary carcinoma, melanotic carcinoma, menigual carcinoma, mesometonephric carcinoma, oat cell carcinoma, squamal cell carcinoma, sweat gland carcinoma, transitional cell carcinoma, and tubular cell carcinoma. Examples of sarcoma include amelioblastic sarcoma, angiolithic sarcoma, botryoid sarcoma, endometrial stroma sarcoma, ewing sarcoma, fascicular sarcoma, giant cell sarcoma, granulocytic sarcoma, immunoblastic sarcoma, juxaccordial osteogenic sarcoma, coppices sarcoma, leukocytic sarcoma (also known as leukemia), lymphatic sarcoma (also known as lympho sarcoma), medullary sarcoma, myeloid sarcoma (also known as granulocytic sarcoma), austiogenci sarcoma, periosteal sarcoma, reticulum cell sarcoma (also known as histiocytic lymphoma), round cell sarcoma, spindle cell sarcoma, synovial sarcoma, and telangiectatic audiogenic sarcoma. Examples of lymphomas include Hodgkin""s disease and lymphocytic lymphomas, such as Burkitt""s, nodular poorly-differentiated lymphocytic (NPDL), nodular mixed lymphocytic (NML), NH (nodular histiocytic), and diffuse lymphomas. Additional carcinomas include neural blastoma, glioblastoma, astrocytoma, melanoma, leiomyo sarcoma, multiple myeloma, and Hemangioma.
A tripeptide aldehyde protease inhibitor (benzyloxycarbonyl (Z)xe2x80x94Leu-Leu-leucinal induces neurite outgrowth in PC12 cells at an optimal concentration of 30 nM, Tsubuki et al., Biochem. and Biophys. Res. Comm. (1993) 196:1195-1201. Peptide aldehydes have been shown to inhibit the chymotryptic activity of the proteasome. Vinitsky, et al., 1992, Tsubuki et al., 1993. One embodiment of the invention is a method of promoting neurite outgrowth, including administering to the subject a compound of a formula disclosed herein.
Finally, the disclosed compounds are also useful as diagnostic agents (e.g., in diagnostic kits or for use in clinical laboratories) for screening for proteins (e.g., enzymes, transcription factors) processed by the proteasome. The disclosed compounds are also useful as research reagents for specifically binding the X/MB1 subunit or xcex1-chain and inhibiting the proteolytic activities associated with it. For example, the activity of (and specific inhibitors of) other subunits of the proteasome can be determined.
Most cellular proteins are subject to proteolytic processing during maturation or activation. Lactacystin can be used to determine whether a cellular, developmental, or physiological process or output is regulated by the proteolytic activity of the proteasome. One such method includes obtaining an organism, an intact cell preparation, or a cell extract; exposing the organism, cell preparation, or cell extract to a compound of a formula disclosed herein; exposing the compound-exposed organism, cell preparation, or cell extract to a signal, and monitoring the process or output. The high selectivity of the compounds disclosed herein permits rapid and accurate elimination or implication of the proteasome in a given cellular, developmental, or physiological process.
The compounds and compositions of the invention are useful in several methods including a method of treating inflammation, comprising administering to a subject an effective anti-inflammatory amount of a pharmaceutical composition described herein (e.g., a lactacystin thioester, a lactone, or a lactam); wherein the inflammation is associated with injury or infection; wherein the inflammation is associated with an allergy or asthma; wherein the inflammation is associated with a disease state selected from rheumatoid arthritis, scleroderma, rheumatic fever, inflammatory bowel disease, diabetes mellitus, myasthenia gravis, multiple sclerosis, Guillan-Barre syndrome, conjunctiva of the eye, systemic lupus erythematosus, encephalitis, Adult Respiratory Distress Syndrome, emphysema, Alzheimer""s disease, and muscular dystrophy; wherein the inflammation is associated with transplantation of bone marrow or a solid organ selected from kidney, lung, heart, pancreas, liver, and skin, and the composition is administered before, during, or after transplantation; wherein the pharmaceutical composition is administered orally; or combinations thereof.
The invention also provides a method of treating cancer, comprising administering to a subject an effective anti-cancer amount of a pharmaceutical composition described herein; a method for treating psoriasis, comprising administering to a subject an effective amount of a pharmaceutical composition described herein; and a method for treating restenosis, comprising administering to a subject an effective amount of a pharmaceutical composition described herein. Preferred compounds and compositions include various lactacystin thioesters, and lactacystin P-lactone analogs, including the p-lactone itself.
Formulation and Administration
The methods of the invention contemplate treatment of animal subjects, such as mammals (e.g., higher primates, and especially humans). The invention encompasses pharmaceutical compositions which include novel compounds described herein, and pharmaceutical compositions which include compounds described and first recognized herein as proteasome inhibitors, such as lactacystin.
Pharmaceutically acceptable salts may be formed, for example, with 1, 2, 3, or more equivalents of hydrogen chloride, hydrogen bromide, trifluoroacetic acid, and others known to those in the art of drug formulation. Compounds of the invention can be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable non-toxic excipients and carriers. A pharmaceutical composition of the invention may contain more than one compound of the invention, and/or may also contain other therapeutic compounds not encompassed by the invention, such as anti-inflammatory, anti-cancer, or other agents. A subject may have more than one type of inflammation, or more than one type of cancer, a combination of allergies, or a mixture of the above conditions for which the disclosed compounds are useful. A compound of the invention may be administered in unit dosage form, and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in Remington""s Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa., 1980). The invention also encompasses a packaged drug, containing a pharmaceutical composition formulated into individual dosages and printed instructions for self-administration.
Compounds disclosed herein as proteasome inhibitors may be prepared for use in parenteral administration in the form of solutions or liquid suspensions; for oral administration (preferable), particularly in the form of tablets or capsules; or intranasally, particularly in the form of powders, gels, oily solutions, nasal drops, aerosols, or mists. Formulations for parenteral administration may contain as common excipients sterile water or sterile saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like. Controlled release of a compound of the invention may be obtained, in part, by use of biocompatible, biodegradable polymers of lactide, and copolymers of lactide/glycolide or polyoxyethylene/polyoxypropylene. Additional parental delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation administration contain lactose, polyoxyethylene-9-lauryl ether, glycocholate, or deoxycholate. Formulations for buccal administration may include glycocholate; formulations for vaginal administration may include citric acid.
The concentration of a disclosed compound in a pharmaceutically acceptable mixture will vary depending on several factors, including the dosage of the compound to be administered, the pharmacokinetic characteristics of the compound(s) employed, and the route of administration. In general, the compounds of this invention may be provided in an aqueous physiological buffer solution containing about 0.1-10% w/v of compound for parenteral administration. Typical dose ranges are from about 0.1 to about 50 mg/kg of body weight per day, given in 1-4 divided doses. Each divided dose may contain the same or different compounds of the invention. The dosage will be an effective amount depending on several factors including the overall health of a patient, and the formulation and route of administration of the selected compound(s).
The effective amount of the active compound used to practice the present invention for treatment of conditions directly or indirectly mediated by the proteasome varies depending upon the manner of administration, the age and the body weight of the subject and the condition of the subject to be treated, and ultimately will be decided by the attending physician or veterinarian. Such amount of the active compound as determined by the attending physician or veterinarian is referred to herein as xe2x80x9ceffective amountxe2x80x9d.
Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. The following specific examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are hereby incorported by reference.